Microrna inhibition for the treatment of inflammation and myeloproliferative disorders

ABSTRACT

The present disclosure relates to the finding that microRNA-155 plays a role in inflammation, hematopoiesis and myeloproliferation, and that dysregulation of microRNA-155 expression is associated with particular myeloproliferative disorders. Disclosed herein are methods and compositions for diagnosing an treating disorders, including inflammation and myeloproliferation, modulating the levels of expression of one or more genes selected from the group consisting of Cutl1, Arntl, Picalm, Jarid2, PU.1, Csf1r, HIF1α, Sla, Cepbβ, and Bach1, and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.Non-provisional application Ser. No. 15/016,096, filed on Feb. 4, 2016,which is a continuation application of U.S. Non-provisional applicationSer. No. 14/138,472, filed on Dec. 23, 2013, now issued as U.S. Pat. No.9,290,761, which is in turn a continuation application of U.S.Non-provisional application Ser. No. 12/122,595, filed on May 16, 2008,now issued as U.S. Pat. No. 8,697,672, which claims the benefit of U.S.Provisional Application Ser. No. 60/930,461, filed on May 16, 2007, byBaltimore et al., and entitled “Inflammatory Cytokines and PathogenAssociated Molecular Patterns Induce Expression of the MammalianOncogene microRNA-155.” The entire disclosure of these relatedapplications is herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under Grant No. GM039458awarded by the National Institutes of Health. The government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledseq_listing_REGLS001C2.TXT, created Feb. 4, 2016, which is 15.2 KB insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in its entirety.

BACKGROUND Field of the Invention

Embodiments disclosed herein relate to molecular medicine, and inparticular to compositions and methods that can be used for diagnosingand treating inflammation and myeloproliferative disorders, among otherthings.

Description of the Related Art

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negativelyregulate their mRNA targets by binding with imperfect complementarity inthe 3′-untranslated region. Largely unrecognized before 2001, it is nowclear that miRNA represent a widely conserved mechanism ofpost-transcriptional gene regulation. In mammals, regulatory roles havebeen identified for miRNA in many areas of biology, pointing to miRNA asan exciting new class of therapeutic targets with broad applications.

SUMMARY OF THE INVENTION

Provided herein are methods of detecting acute myeloid leukemia in asubject, comprising identifying a subject suspected of having acutemyeloid leukemia, measuring the levels of microRNA-155 (miR-155) in thebone marrow of said subject, and identifying the subject as having acutemyeloid leukemia (AML) if said levels of miR-155 are elevated comparedto control levels. In certain embodiments, the control levels can be themiR-155 levels in the bone marrow of a subject that does not have AML orother myeloproliferative disorder. In certain embodiments, the controllevels can be the average levels of miR-155 in a population of subjectsthat do not have AML or other myeloproliferative disorder. In certainembodiments, the control can be the average miR-155 level of apopulation of subjects that do not have AML or other myeloproliferativedisorder. In certain embodiments, the AML is acute myelomonocyticleukemia. In certain embodiments, the AML is acute monocytic leukemia.In certain embodiments, the measuring step comprises performingquantitative PCR. In certain embodiments, the measuring step comprisesperforming a microarray analysis.

Provided herein are methods of treating or preventing an miR-155associated condition, such as a myeloproliferative disorder orinflammation. Provided herein are methods of treating or preventing amyeloproliferative disorder, comprising identifying a subject having, orsuspected of having a myeloproliferative disorder, and administering tosaid subject an miR-155 antagonist. In certain embodiments, themyeloproliferative disorder is acute myeloid leukemia. In certainembodiments, the acute myeloid leukemia is acute myelomonocyticleukemia. In certain embodiments, the acute myeloid leukemia is acutemonocytic leukemia.

Also provided herein are methods of treating inflammation in a subjectin need thereof, comprising identifying a subject in need of a reductionin inflammation and administering to said subject an miR-155 antagonist.In certain embodiments, the inflammation is mediated by a Toll-likereceptor (TLR). In certain embodiments, the TLR may be TLR2, TLR3, TLR,4 or TLR9.

Methods of modulating the expression of one or more miR-155 target genesrelated hematopoiesis and/or myeloproliferation are also providedherein. The gene may be Cutl1, Arntl, Picalm, Jarid2, PU.1, Csf1r,HIF1α, Sla, Cepbβ, or Bach1. The modulation may be achieved bycontacting a target cell with a miR-155 antagonist. The target cell maybe a hematopoietic stem cell, a bone marrow cell, a myeloid precursorcell, and a myeloid cell. The myeloid cell may be a monocyte, amacrophage, or a neutrophil. In certain embodiments, the modulationcomprises increasing expression of one or more of the target genes.

Further provided herein are methods for modulating cell proliferation ina granulocyte/monocyte population comprising contacting thegranulocyte/monocyte population with a miR-155 antagonist. In certainembodiments, cell proliferation is inhibited. In certain embodiments,the rate of cell proliferation is reduced, i.e. cell proliferation isslowed.

Also provided herein are methods for modulating proliferation ofhematopoietic cells comprising contacting the hematopoietic cells with amiR-155 antagonist. In certain embodiments, cell proliferation isinhibited. In certain embodiments, the rate of cell proliferation isreduced, i.e., cell proliferation is slowed. In certain embodiments, thehematopoietic cell is a myeloid cell.

In certain embodiments, the miR-155 antagonist comprises an miR-155antisense compound. In certain embodiments, the miR-155 antisensecompound comprises a modified oligonucleotide consisting of 12 to 30linked nucleosides, wherein the nucleobase sequence of the modifiedoligonucleotide is complementary to a sequence at least 80% identical tomature microRNA-155 (SEQ ID NO: 72), pre-microRNA-155 (SEQ ID NO: 73),or a microRNA-155 seed sequence (SEQ ID NOs: 43-56). In certainembodiments, the nucleobase sequence of the miR-155 oligonucleotide hasno more than two mismatches to the nucleobase sequence of mature miR-155(SEQ ID NO: 72), or pre-miR-155 (SEQ ID NO: 73). In certain embodiments,the modified oligonucleotide is conjugated to a ligand. In certainembodiments, at least one internucleoside linkage of the modifiedoligonucleotide is a modified internucleoside linkage. In certainembodiments, at least one nucleoside of the modified oligonucleotidecomprises a modified sugar. In certain embodiments, each nucleoside ofthe modified oligonucleotide comprises a modified sugar. In certainembodiments, at least one nucleoside of the modified oligonucleotidecomprises a modified nucleobase. In certain embodiments, eachinternucleoside linkage of the modified oligonucleotide comprises amodified internucleoside linkage. In certain embodiments, theinternucleoside linkage is a phosphorothioate internucleoside linkage.In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide comprises at least 15, 16, 17, 18, 19, 20, or 21consecutive nucleobases of SEQ ID NO: 74. In certain embodiments, thenucleobase sequence of the modified oligonucleotide consists of thenucleobase sequence of SEQ ID NO: 74.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are scatter plots showing log₁₀-transformed signalintensities reflecting the amount of microRNA's in wild-type murinemacrophages stimulated with media (FIGS. 1A and 1B, Cy3), or stimulatedwith IFNβ (FIG. 1A, Cy5) or Poly (I:C) (FIG. 1B, Cy3), assessed using amicroarray of miRNA's. miR-155 signals are indicated in each plot.

FIG. 2A is a graph showing the relative expression of miRNA-155 inwild-type murine macrophages stimulated with media alone (m), IFNβ, orpoly (I:C), as measured by quantitative PCR.

FIG. 2B is an image of a Northern Blot probing for miRNA-155 inwild-type murine macrophages stimulated with media (m), IFNβ, or poly(I:C).

FIG. 2C is an image of the gel used for the Northern Blot shown in FIG.2B. Shown is the band corresponding to tRNA for each of the murinemacrophage cultures, media (m), IFNβ, and poly (I:C).

FIGS. 3A-3B are graphs showing the relative expression of U6 (FIG. 3A)and IP10 (FIG. 3B) mRNA's in wild-type murine macrophages stimulatedwith media (m), IFNβ, or poly (I:C), as measured by quantitative PCR.

FIGS. 4A-4C are plots of fluorescence activated cell sorting (FACS).FIG. 4A shows the distribution of wild-type murine macrophagesexpressing cell surface markers CD11b and CD86 following stimulationwith culture medium. FIG. 4B shows the distribution of wild-type murinemacrophages expressing cell surface markers CD11b and CD86 followingstimulation with IFNβ. FIG. 4B shows the distribution of wild-typemurine macrophages expressing cell surface markers CD11b and CD86following stimulation with poly (I:C).

FIG. 5A is a graphical depiction of the human BIC non-coding RNA geneshowing the location of miR-155 in exon 3. “E” represents “exon”, “I”represents “intron”.

FIG. 5B is an image of an agarose gel showing BIC mRNA levels inwild-type murine macrophage cells treated with media supplemented withIFNβ or poly (I:C), as measured by RT-PCR, at indicated time pointsfollowing treatment. Levels of L32 RNA prepared from mRNA from the samecultures are also shown.

FIG. 5C is a graph showing the relative expression levels (logarithmicscale) of miR-155 over time in wild-type murine macrophage cells treatedwith media (m), IFNβ, or poly (I:C), as measured by quantitative PCR.

FIG. 6A is an image of a Northern Blot of RNA isolated from wild-typemurine macrophages stimulated with media (m), poly (I:C), LPS, CpG, orPam3CSK4 (P3C), using probes for miR-155. The bands corresponding tomature miR-155 and miR-155 are shown. Also shown in FIG. 6B is an imageof the agarose gel used in the Northern Blotting experiments, stainedwith ethidium bromide. The band corresponding to tRNA is shown on theagarose gel.

FIG. 7A is a graph showing relative expression of miR-155 in wild-type,MyD88^(−/−), and TRIF^(−/−) murine macrophages stimulated with medium,poly (I:C), LPS, CpG, or Pam3CSK4 (P3C), as measured by quantitativePCR.

FIG. 7B is a graph showing relative expression of miR-155 in wild-typeand IFNAR^(−/−) murine macrophages stimulated with medium, poly (I:C),LPS, CpG, or Pam3CSK4 (P3C), as measured by quantitative PCR.

FIG. 8A is a graph showing relative expression of miR-155 in wild-typeand TNFR^(−/−) murine macrophages stimulated with medium, IFNβ, or IFNγas measured by quantitative PCR.

FIG. 8B is a graph showing relative expression of miR-155 in wild-typeand TNFR^(−/−) murine macrophages stimulated with medium, or TNFα asmeasured by quantitative PCR.

FIG. 8C is a graph showing relative expression of TNFα in wild-type andTNFR^(−/−) murine macrophages stimulated with medium, IFNβ, or IFNγ asmeasured by quantitative PCR.

FIG. 8D is a graph showing relative expression of IP10 in wild-type andTNFR^(−/−) murine macrophages stimulated with medium or IFNβ, asmeasured by quantitative PCR.

FIG. 8E is a graph showing relative expression of TNFα in wild-type andTNFR^(−/−) murine macrophages stimulated with medium or poly (I:C) asmeasured by quantitative PCR.

FIG. 8F is a graph showing relative expression of miR-155 in wild-typeand TNFR^(−/−) murine macrophages stimulated with medium or poly (I:C)as measured by quantitative PCR.

FIGS. 9A-9B are graphs of the relative expression of miR-155 inwild-type murine macrophages pretreated with DMSO or sp6000125, andsubsequently stimulated with medium, poly (I:C) (FIG. 9A) or TNFα (FIG.9B).

FIG. 10 is a graph showing the relative expression of miR-155 over timein wild-type murine macrophages stimulated with TNFa.

FIG. 11A is a graph showing the relative expression of miR-155 in bonemarrow cells of wild-type mice injected with either phosphate bufferedsaline (PBS) or LPS, at 24 and 72 hours post-injection, as measured byquantitative PCR.

FIG. 11B is a graph showing the relative expression of miR-155 in bonemarrow cells from wild-type or Rag^(−/−) mice, following in vitrostimulation with LPS, GM-CSF (GM), or medium (M), as measured byquantitative PCR.

FIGS. 12A-12F are plots of fluorescence activated cell sorting (FACS).

FIGS. 12A and 12B show the distribution of bone marrow cells isolatedfrom mice that express cell surface markers B220 and Mac1, 72 hoursafter the mice were injected with phosphate buffered saline (PBS) (FIG.12A) or LPS (FIG. 12B). FIGS. 12C and 12D show the distribution of bonemarrow cells isolated from mice that express Gr1 and Mac1, 72 hoursafter the mice were injected with PBS (FIG. 12C) or LPS (FIG. 12D).FIGS. 12E and 12F show the distribution of bone marrow cells isolatedfrom mice that express CD4 and Ter-119, 72 hours after the mice wereinjected with PBS (FIG. 12E) or LPS (FIG. 12F).

FIGS. 13A-13D are photographs of bone marrow cells isolated from miceinjected with PBS and stained with hematoxylin and eosin (H&E) (FIG.13A) or Wright's stain (FIG. 13B), or of bone marrow cells isolated frommice injected with LPS and stained with H&E (FIG. 13C) or Wright's stain(FIG. 13D).

FIG. 14A is a graphical representation (not to scale) of the expressionvector used to transform hematopoietic stem cells (HSC's) to induceexpression of an miR-155-GFP fusion protein, MG155.

FIGS. 14B-14C are plots of fluorescence activated cell sorting (FACS).

FIGS. 14B and 14C show the distribution GFP⁺ bone marrow cells isolatedfrom mice reconstituted with HSC's transformed with a control vector(Cont, FIG. 14B), or an miR-155 expression vector MG155 (MG155, FIG.14C) compared to control (non-reconstituted) mice.

FIG. 14D is a graph showing the expression of MG155 in the bone marrowof mice reconstituted with MG155, or a control vector, as measured byFACS. B6 corresponds to untreated mice, C corresponds to micereconstituted with HSC's transformed with the control vector, and MG155corresponds to mice reconstituted with the MG155 transformed HSC's.

FIG. 15 is a photograph showing the tibias from mice reconstituted withHSC's transformed with MG155 (MG155) or control vector (Cont), twomonths following reconstitution, compared to untreated mice (B6).

FIGS. 16A-16D are photographs of Hematoxylin and Eosin stained bonemarrow sections from mice reconstituted with miR-155 expressing HSC's(FIG. 16A) compared to control mice (FIG. 16B); and Wright's stainedbone marrow sections from mice reconstituted with miR-155 expressingHSC's (FIG. 16C) compared to control mice (FIG. 16D).

FIG. 17A-17D are photographs of dysplastic myeloid cells observed inmiR-155-expressing bone marrow.

FIG. 17E is a bar graph showing the number of cells expressing Mac1/GR1;Ter-119, CD4 or B220 cell surface markers in the bone marrow of controlmice (grey) or mice reconstituted with HSC's transformed with MG155(black). The bar indicates 25 μm.

FIGS. 18A-18D are plots showing the distribution of GFP-gated cellsexpressing SSC and FSC cell surface markers in control mice (FIG. 18A)or mice reconstituted with MG155 HSC's (FIG. 18B); and the distributionof cells expressing Gr1 and Mac1 cell surface markers in control mice(FIG. 18C) or mice reconstituted with MG155 HSC's (FIG. 18D).

FIGS. 19A-19F are FACS plots showing the distribution of bone marrowcells from mice reconstituted with HSC's transformed with MG155 orcontrol vector. Shown is the distribution of cells expressing Mac1(FIGS. 19A, 19B), Ter-119 (FIGS. 19C, 19D), and B220 (FIGS. 19E, 19F) onboth GFP⁺ and GFP⁻ cells.

FIG. 20A is a photograph of a spleen removed from mice reconstitutedwith HSC's transformed with MG155 or control vector, two months afterreconstitution.

FIG. 20B is a bar graph of the average spleen weight of micereconstituted with HSC's transformed with MG155 or control vector twomonths after reconstitution.

FIGS. 21A-21D are photographs of hematoxylin & eosin (H&E) stainedsections (FIGS. 21A, 21B) or Wright's stained sections (FIGS. 21C, 21D)from spleens of mice reconstituted with HSC's transformed with MG155 orcontrol vector. The bar indicates 25 μm.

FIG. 22 is a bar graph of the average number of cells expressingMac1/GR1, Ter-199, CD4 and B220 cell surface markers in micereconstituted with HSC's transformed with MG155 or control vector.

FIGS. 23A-23D are plots showing the cell distribution of GFP-gatedspleen cells from mice reconstituted with MG155 (FIGS. 23B, 23D) orcontrol vector HSC's (FIGS. 23A, 23C), assessing SSC and FSC, orGr1/Mac1 cell surface markers, as measured by FACS.

FIGS. 24A-24H are plots showing the distribution of both GFP+ andGFP-splenocytes from mice reconstituted with control vector or MG155HSC's. The plots show the distribution of cells expressing the cellsurface markers Mac1 (FIGS. 24A, 24B), Ter-119 (FIGS. 24C, 24D), CD4(FIGS. 24E, 24F), and B220 (FIGS. 24G, 24H).

FIGS. 25A-25D are plots showing the distribution of peripheral bloodcells expressing SSC, FSC, or SSC and Mac1, in mice reconstituted withcontrol vector or MG155, two months after reconstitution.

FIG. 25E is a graph showing the total number of Mac1-expressingperipheral blood cells (K/μl)

FIGS. 26A-26D are photomicrographs of a normal Wright's-stained monocyte(Mo) (FIG. 26A, 26B) and neutrophil (Ne) (FIG. 26C, FIG. 26D) from theblood of mice reconstituted with control vector HSC's or MG155 HSC's.

FIG. 27A-27F are graphs showing the levels of red blood cells (RBC's)(FIG. 27A), white blood cells (WBC's) (FIG. 27B), hemoglobin-expressingcells (Hb) (FIG. 27C), B220 B cells (B220) (FIG. 2D), platelet (PLT)(FIG. 27E), and CD4 T cell levels (FIG. 27F) in the blood of micereconstituted with MG155 (155) or control vector (c) HSC's.

FIGS. 28A-28B are photographs of Wright-stained peripheral blood redblood cells from mice reconstituted with control vector (FIG. 28A) orMG155 (FIG. 28B) HSC's, two months after reconstitution. The barrepresents 10 μm.

FIGS. 29A-29B are graphs showing the relative levels of miR-155RNA (FIG.29A) and 5S RNA (FIG. 29B) in bone marrow cells of normal subjects(Norm) or subjects diagnosed with acute myeloid lymphoma (AML). Levelsare expressed as fold above background (log scale).

FIGS. 29C-29D are graphs showing the relative levels of miR-155 RNA(FIG. 29C) and 5S RNA (FIG. 29D) in bone marrow cells of normal subjects(Norm) or subjects diagnosed with acute myeloid lymphoma of the FABsubtype M4 or M5.

FIG. 30 is a bar graph showing the percent mRNA expression of theindicated genes in Raw 264.7 cells infected with MSCVpuro-155 or emptyvector, as analyzed by micro-array and quantitative PCR. Numericalrepression values for each mRNA are listed. For both the array and qPCRdata, all values were normalized to L32 RNA levels, are displayed as thepercent expression of control, and are the average of three independentexperiments.

FIG. 31A is an image of a Western Blot to assay Cebpb, PU.1 cut1, Picalmand α-Tubulin in Raw 264.7 cells stably expressing miR-155 (155) orempty vector (c).

FIG. 31B is a bar graph showing the % protein expression of control ofCebpb, PU.1, Cutl1, Picalm and Tubulin in Raw 264.7 cells stablyexpressing miR-155.

FIG. 31C is an image of a Northern Blot probing for miR-155, using RNAisolated from Raw 264.7 cells stably expressing miR-155 (155) or emptyvector (−).

FIG. 32 shows a graphical depiction of the location of conserved miR-155sites in 3′ untranslated regions (3′ UTRs) of the indicated genes. Greyboxes denote conserved 7 or 8-mer seeds, white boxes denotenon-conserved 7-mer seeds, or conserved 6-mer seeds. The X's throughboxes indicate mutation s to seed regions. The line above the shown 3′UTR's indicates regions that were cloned downstream of from a luciferasereporter gene. Shown to the right is the % luciferase expression ofcontrol in 293T cells co-transformed with the indicated luciferasereporter constructs and a β-galactosidase expression plasmid, and anmiR-155 expression vector or empty vector control. Data using wild-type3′UTR's are in black. Data using mutant 3′ UTR's are shown in gray. Dataare a triplicate set representing three independent experiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

“Subject” means a human or non-human animal selected for treatment ortherapy.

“Subject suspected of having” means a subject exhibiting one or moreclinical indicators of a disease or condition. In certain embodiments,the disease or condition is inflammation. In certain embodiments, thedisease or condition is a myeloproliferative disorder.

“Subject suspected of having inflammation” means a subject exhibitingone or more clinical indicators of inflammation.

“Subject suspected of having a myeloproliferative disorder” means asubject exhibiting one or more clinical indicators of amyeloproliferative disorder.

“Preventing” or “prevention” refers to delaying or forestalling theonset, development or progression of a condition or disease for a periodof time, including weeks, months, or years.

“Treatment” or “treat” means the application of one or more specificprocedures used for the cure or amelioration of a disease. In certainembodiments, the specific procedure is the administration of one or morepharmaceutical agents.

“Amelioration” means a lessening of severity of at least one indicatorof a condition or disease. In certain embodiments, amelioration includesa delay or slowing in the progression of one or more indicators of acondition or disease. The severity of indicators may be determined bysubjective or objective measures which are known to those skilled in theart.

“Subject in need thereof” means a subject identified as in need of atherapy or treatment.

“Administering” means providing a pharmaceutical agent or composition toa subject, and includes, but is not limited to, administering by amedical professional and self-administering.

“Parenteral administration,” means administration through injection orinfusion.

Parenteral administration includes, but is not limited to, subcutaneousadministration, intravenous administration, intramuscularadministration, intraarterial administration, and intracranialadministration.

“Subcutaneous administration” means administration just below the skin.

“Intravenous administration” means administration into a vein.

“Intraarterial administration” means administration into an artery.

“Improves liver function” means the changes liver function toward normalparameters. In certain embodiments, liver function is assessed bymeasuring molecules found in a subject's blood. For example, in certainembodiments, improved liver function is measured by a reduction in bloodliver transaminase levels.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual that includes a pharmaceutical agent. Forexample, a pharmaceutical composition may comprise a modifiedoligonucleotide and a sterile aqueous solution.

“Pharmaceutical agent” means a substance that provides a therapeuticeffect when administered to a subject.

“Active pharmaceutical ingredient” means the substance in apharmaceutical composition that provides a desired effect.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and“nucleic acid target” all mean a nucleic acid capable of being targetedby antisense compounds.

“Targeting” means the process of design and selection of nucleobasesequence that will hybridize to a target nucleic acid and induce adesired effect.

“Targeted to” means having a nucleobase sequence that will allowhybridization to a target nucleic acid to induce a desired effect. Incertain embodiments, a desired effect is reduction of a target nucleicacid.

“Modulation” means to a perturbation of function or activity. In certainembodiments, modulation means an increase in gene expression. In certainembodiments, modulation means a decrease in gene expression.

“Expression” means any functions and steps by which a gene's codedinformation is converted into structures present and operating in acell.

“5′ target site” refers to the nucleobase of a target nucleic acid whichis complementary to the 5′-most nucleobase of a particularoligonucleotide.

“3′ target site” means the nucleobase of a target nucleic acid which iscomplementary to the 3′-most nucleobase of a particular oligonucleotide.

“Region” means a portion of linked nucleosides within a nucleic acid. Incertain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of a target nucleic acid. Forexample, in certain such embodiments a modified oligonucleotide iscomplementary to a region of a miRNA stem-loop sequence. In certain suchembodiments, a modified oligonucleotide is 100% identical to a region ofa miRNA sequence.

“Segment” means a smaller or sub-portion of a region.

“Nucleobase sequence” means the order of contiguous nucleobases, in a 5′to 3′ orientation, independent of any sugar, linkage, and/or nucleobasemodification.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother in a nucleic acid.

“Nucleobase complementarity” means the ability of two nucleobases topair non-covalently via hydrogen bonding.

“Complementary” means a first nucleobase sequence is at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical, or is 100%identical, to the complement of a second nucleobase sequence over aregion of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ormore nucleobases, or that the two sequences hybridize under stringenthybridization conditions. In certain embodiments a modifiedoligonucleotide that has a nucleobase sequence which is 100%complementary to a miRNA, or precursor thereof, may not be 100%complementary to the miRNA, or precursor thereof, over the entire lengthof the modified oligonucleotide.

“Complementarity” means the nucleobase pairing ability between a firstnucleic acid and a second nucleic acid.

“Full-length complementarity” means each nucleobase of a first nucleicacid is capable of pairing with each nucleobase at a correspondingposition in a second nucleic acid. For example, in certain embodiments,a modified oligonucleotide wherein each nucleobase has complementarityto a nucleobase in an miRNA has full-length complementarity to themiRNA.

“Percent complementary” means the number of complementary nucleobases ina nucleic acid divided by the length of the nucleic acid. In certainembodiments, percent complementarity of a modified oligonucleotide meansthe number of nucleobases that are complementary to the target nucleicacid, divided by the number of nucleobases of the modifiedoligonucleotide. In certain embodiments, percent complementarity of amodified oligonucleotide means the number of nucleobases that arecomplementary to a miRNA, divided by the number of nucleobases of themodified oligonucleotide.

“Percent region bound” means the percent of a region complementary to anoligonucleotide region. Percent region bound is calculated by dividingthe number of nucleobases of the target region that are complementary tothe oligonucleotide by the length of the target region. In certainembodiments, percent region bound is at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%.

“Percent identity” means the number of nucleobases in first nucleic acidthat are identical to nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

“Substantially identical” used herein may mean that a first and secondnucleobase sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98% or 99% identical, or 100% identical, over a region of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases.

“Hybridize” means the annealing of complementary nucleic acids thatoccurs through nucleobase complementarity.

“Mismatch” means a nucleobase of a first nucleic acid that is notcapable of pairing with a nucleobase at a corresponding position of asecond nucleic acid.

“Non-complementary nucleobase” means two nucleobases that are notcapable of pairing through hydrogen bonding.

“Identical” means having the same nucleobase sequence.

“miRNA” or “miR” means a non-coding RNA between 18 and 25 nucleobases inlength which hybridizes to and regulates the expression of a coding RNA.In certain embodiments, a miRNA is the product of cleavage of apre-miRNA by the enzyme Dicer. Examples of miRNAs are found in the miRNAdatabase known as miRBase (http://microrna.sanger.ac.uk/).

“Pre-miRNA” or “pre-miR” means a non-coding RNA having a hairpinstructure, which contains a miRNA. In certain embodiments, a pre-miRNAis the product of cleavage of a pri-miR by the double-strandedRNA-specific ribonuclease known as Drosha.

“Stem-loop sequence” means an RNA having a hairpin structure andcontaining a mature miRNA sequence. Pre-miRNA sequences and stem-loopsequences may overlap. Examples of stem-loop sequences are found in themiRNA database known as miRBase (http://microrna.sanger.ac.uk/).

“Pri-miRNA” or “pri-miR” means a non-coding RNA having a hairpinstructure that is a substrate for the double-stranded RNA-specificribonuclease Drosha.

“miRNA precursor” means a transcript that originates from a genomic DNAand that comprises a non-coding, structured RNA comprising one or moremiRNA sequences. For example, in certain embodiments a miRNA precursoris a pre-miRNA. In certain embodiments, a miRNA precursor is apri-miRNA.

“Monocistronic transcript” means a miRNA precursor containing a singlemiRNA sequence.

“Polycistronic transcript” means a miRNA precursor containing two ormore miRNA sequences.

“Seed region” means nucleotides 2 to 6 or 2 to 7 from the 5′-end of amature miRNA sequence.

“Oligomeric compound” means a compound comprising a polymer of linkedmonomeric subunits.

“Antisense compound” means a compound having a nucleobase sequence thatwill allow hybridization to a target nucleic acid. In certainembodiments, an antisense compound is an oligonucleotide having anucleobase sequence complementary to a target nucleic acid.

“Oligonucleotide” means a polymer of linked nucleosides, each of whichcan be modified or unmodified, independent from one another.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage between nucleosides.

“Natural sugar” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Natural nucleobase” means a nucleobase that is unmodified relative toits naturally occurring form.

“Internucleoside linkage” means a covalent linkage between adjacentnucleosides.

“Linked nucleosides” means nucleosides joined by a covalent linkage.

“Nucleobase” means a heterocyclic moiety capable of non-covalentlypairing with another nucleobase.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of a nucleoside.

“miR antagonist” means an agent designed to interfere with or inhibitthe activity of a miRNA. In certain embodiments, a miR antagonistcomprises an antisense compound targeted to a miRNA. In certainembodiments, a miR antagonist comprises a modified oligonucleotidehaving a nucleobase sequence that is complementary to the nucleobasesequence of a miRNA, or a precursor thereof. In certain embodiments, amiR antagonist is a miR-155 antagonist. In other embodiments, an miR-155antagonist comprises a small molecule, or the like that interferes withor inhibits the activity of an miRNA.

“miR-155 antagonist” means an agent designed to interfere with orinhibit the activity of miR-155.

“Modified oligonucleotide” means an oligonucleotide having one or moremodifications relative to a naturally occurring terminus, sugar,nucleobase, and/or internucleoside linkage.

“Single-stranded modified oligonucleotide” means a modifiedoligonucleotide which is not hybridized to a complementary strand.

“Modified internucleoside linkage” means any change from a naturallyoccurring internucleoside linkage.

“Phosphorothioate internucleoside linkage” means a linkage betweennucleosides where one of the non-bridging atoms is a sulfur atom.

“Modified sugar” means substitution and/or any change from a naturalsugar.

“Modified nucleobase” means any substitution and/or change from anatural nucleobase.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position.

“2′-O-methyl sugar” or “2′-OMe sugar” means a sugar having a O-methylmodification at the 2′ position.

“2′-O-methoxyethyl sugar” or “2′-MOE sugar” means a sugar having a0-methoxyethyl modification at the 2′ position.

“2′-O-fluoro sugar” or “2′-F sugar” means a sugar having a fluoromodification of the 2′ position.

“Bicyclic sugar moiety” means a sugar modified by the bridging of twonon-geminal ring atoms.

“2′-O-methoxyethyl nucleoside” means a 2′-modified nucleoside having a2′-O-methoxyethyl sugar modification.

“2′-fluoro nucleoside” means a 2′-modified nucleoside having a 2′-fluorosugar modification.

“2′-O-methyl” nucleoside means a 2′-modified nucleoside having a2′-O-methyl sugar modification.

“Bicyclic nucleoside” means a 2′-modified nucleoside having a bicyclicsugar moiety.

“Motif” means a pattern of modified and/or unmodified nucleobases,sugars, and/or internucleoside linkages in an oligonucleotide.

A “fully modified oligonucleotide” means each nucleobase, each sugar,and/or each internucleoside linkage is modified.

A “uniformly modified oligonucleotide” means each nucleobase, eachsugar, and/or each internucleoside linkage has the same modificationthroughout the modified oligonucleotide.

A “stabilizing modification” means a modification to a nucleoside thatprovides enhanced stability to a modified oligonucleotide, in thepresence of nucleases, relative to that provided by 2′-deoxynucleosideslinked by phosphodiester internucleoside linkages. For example, incertain embodiments, a stabilizing modification is a stabilizingnucleoside modification. In certain embodiments, a stabilizingmodification is a internucleoside linkage modification.

A “stabilizing nucleoside” means a nucleoside modified to provideenhanced nuclease stability to an oligonucleotide, relative to thatprovided by a 2′-deoxynucleoside. In one embodiment, a stabilizingnucleoside is a 2′-modified nucleoside.

A “stabilizing internucleoside linkage” means an internucleoside linkagethat provides enhanced nuclease stability to an oligonucleotide relativeto that provided by a phosphodiester internucleoside linkage. In oneembodiment, a stabilizing internucleoside linkage is a phosphorothioateinternucleoside linkage.

Certain Diseases, Conditions, and Cellular Phenotypes

MiR-155 is strongly induced in cells of the innate immune system afterexposure to inflammatory stimuli. As described herein, miR-155expression in bone marrow-derived macrophages is increased by severalToll-like receptor (TLR) ligands through MyD88 or TRIF signaling and byinterferons through TNF-alpha autocrine or paracrine signaling. Thus,miR-155 is a common target of a broad range of inflammatory mediators.

The inflammatory response to infection must be carefully regulated toachieve pathway clearance and prevent the consequences of unrelated geneexpression. The inflammatory process is known to have a significantimpact on the generation of blood cells from a common hematopoietic stemcell (hematopoiesis) by enhancing the production of granulocyte/monocyte(GM) populations to replenish the cells that become depleted whilefighting infection. However, dysregulation of hematopoiesis can lead toexcess proliferation of cells, which can in turn lead to various typesof malignancies. As described herein, miR-155 expression in bone marrowis induced following exposure to inflammatory stimuli and is correlatedwith granulocyte/monocyte (GM) expansion. The sustained expression ofmiR-155 in bone marrow led to profound myeloid proliferation withdysplastic changes, as evidenced by the miR-155-induced GM population ofcells displaying pathological features characteristic of myeloidneoplasia (i.e., a myeloproliferative disorder). A comparison of bonemarrow from patients with certain subtypes of acute myeloid leukemia(AML) revealed that miR-155 is overexpressed in the bone marrow of thesepatients, relative to bone marrow samples of healthy donors. Thus, it isdemonstrated herein that miR-155 contributes to physiological GMexpansion during inflammation and to certain pathological featuresassociated with AML. Accordingly, miR-155 is implicated as a linkbetween the inflammatory response and cancer. Further, miR-155 levelscan be used in the diagnosis and/or classification of certainmyeloproliferative disorders such as AML. For example, miR-155 can beused in the diagnosis and identification of FAB-ALM-M4 and FAB-AML-M5.The data provided herein demonstrate the importance of proper regulationof miR-155 in developing myeloid cells during the inflammatory response,to avoid excessive activation of the inflammatory response and/or thedevelopment of cancer.

Accordingly, miR-155 can be regulated to modulate the innate immunesystem, for example, in the treatment, prevention or amelioration ofdiseases characterized by activation, particularly excessive activation,of the innate immune system. In certain embodiments, treatment isprovided to a subject suffering from inflammation, orinflammatory-related conditions, such as inflammation arising from amacrophage-induced inflammatory response, mediated through a Toll-likeReceptor(s) (TLRs). The inflammation can arise as a result of activationof TLR2, TLR3, TLR4, TLR9, pathways, or the like, for example caused bycancer, viral infection, microbial infection or the like, as describedherein. Additional inflammatory-related conditions include, for example,sepsis and septic shock, neurodegeneration, neutrophilic alveolitis,asthma, hepatitis, inflammatory bowel disease, ischemia/reperfusion,septic shock, glomerulonephritis, rheumatoid arthritis, or Crohn'sdisease. The administration of miR-155 antagonists, such as antisenseoligonucleotides, can be used to interfere with or inhibit the activityof miR-155 and thus inhibit or attenuate inflammation.

Further, miR-155 can be regulated to treat, prevent or amelioratemyeloproliferative disorders. Such myeloproliferative disorders include,without limitation, acute myeloid leukemia. In certain embodiments, theacute myeloid leukemia is acute myelomonocytic leukemia or acutemonocytic leukemia. MiR-155 antagonists, such as a modifiedoligonucleotide having a nucleobase sequence complementary to miR-155,can be used to interfere with or inhibit the activity of miR-155, thusinhibiting the excessive proliferation of myeloid cells.

Microarray analyses were employed to identify target genes that aremodulated by miR-155. These studies revealed that miR-155 can directlyregulate several genes relevant to hematopoiesis and myeloproliferation,including but not limited to Bach1, PU.1, Cutl1, Picalm, Arntl, Csf1r,Sla, Jarid2, and Hlfla. Accordingly, miR-155 antagonists can be used tomodulate the expression of genes involved in hematopoiesis andmyeloproliferation. MiR-155 antagonists, such as a modifiedoligonucleotide having a nucleobase sequence complementary to miR-155,can be delivered to myeloid cells to modulate the expression of miR-155target genes.

Antagonists of miR-155 can also be used to slow, prevent or inhibit theproliferation of cells in a granulocyte/monocyte cell population. Incertain embodiments, the granulocyte/monocyte cell expansion occursduring the inflammatory response.

Additionally, antagonists of miR-155 can be used to slow, prevent orinhibit hematopoietic cell proliferation. In certain embodiments, thehematopoietic cell proliferation comprises myeloid cell proliferation.In certain embodiments, the myeloid cell proliferation is associatedwith the inflammatory response. In certain embodiments, the myeloid cellproliferation is associated with a myeloproliferative disorder, such asacute myeloid leukemia.

As illustrated herein, elevated miR-155 levels are associated withinflammation. As such, provided herein are methods of detectingmiR-155-mediated inflammation in a subject, comprising measuring miR-155levels in the cells of a subject suspected of having miR-155-mediatedinflammation. As further illustrated herein, elevated miR-155 levels areobserved in connection with myeloid cell proliferation. Accordingly,provided herein are methods of detecting a miR-155 mediatedmyeloproliferative disorder in a subject, comprising measuring miR-155levels in the bone marrow of a subject suspected of having amiR-155-mediated myeloproliferative disorder.

Certain Routes of Administration

In certain embodiments, administering to a subject comprises parenteraladministration. In certain embodiments, administering to a subjectcomprises intravenous administration. In certain embodiments,administering to a subject comprises subcutaneous administration.

In certain embodiments, administration includes pulmonaryadministration. In certain embodiments, pulmonary administrationcomprises delivery of aerosolized oligonucleotide to the lung of asubject by inhalation. Following inhalation by a subject of aerosolizedoligonucleotide, oligonucleotide distributes to cells of both normal andinflamed lung tissue, including alveolar macrophages, eosinophils,epithelium, blood vessel endothelium, and bronchiolar epithelium. Asuitable device for the delivery of a pharmaceutical compositioncomprising a modified oligonucleotide includes, but is not limited to, astandard nebulizer device. Formulations and methods for modulating thesize of droplets using nebulizer devices to target specific portions ofthe respiratory tract and lungs are well known to those skilled in theart. Additional suitable devices include dry powder inhalers or metereddose inhalers.

In certain embodiments, pharmaceutical compositions are administered toachieve local rather than systemic exposures. For example, pulmonaryadministration delivers a pharmaceutical composition to the lung, withminimal systemic exposure.

Additional suitable administration routes include, but are not limitedto, oral, rectal, transmucosal, intestinal, enteral, topical,suppository, intrathecal, intraventricular, intraperitoneal, intranasal,intraocular, intramuscular, intramedullary, and intratumoral.

Certain Additional Therapies

Treatments for disorders listed herein may comprise more than onetherapy. As such, in certain embodiments provided herein are methods fortreating a subject having or suspected of having an inflammatorydisorder described herein comprising administering at least one therapyin addition to administering a miR-155 antagonist. In certainembodiments, provided herein are methods for treating a subject havingor suspected of having a myeloproliferative disorder described hereincomprising administering at least one therapy in addition toadministering a miR-155 antagonist.

In certain embodiments, the at least one additional therapy comprises achemotherapeutic agent. In certain embodiments, chemotherapeutic agentsinclude, but are not limited to, cytarabine (also known as cytosinearabinoside or ara-C(Cytosar)), daunorubicin (also known as daunomycin(Cerubidine), idarubicin (e.g., Idamycin), mitoxantrone (e.g.,Novantrone), 6-thioguanine (also known as 6-TG), 6-mercaptopurine, (alsoknown as 6-MP; e.g., Purinethol), fludarabine (e.g., Fludara),vincristine (e.g., Oncovin), and etoposide (e.g., VePesid, others).

In certain embodiments, the at least one additional therapy comprises animmunosuppressant. In certain embodiments, an immunosuppressantincludes, but is not limited to, a glucocorticoid, a cytostatic, and anantibody. In certain embodiments, an immunosuppressant is acorticosteroid, such as prednisone.

In certain embodiments, the methods provided herein compriseadministering one or more additional pharmaceutical agents. In certainembodiments, additional pharmaceutical agents include, but are notlimited to, diuretics (e.g. sprionolactone, eplerenone, furosemide),inotropes (e.g. dobutamine, milrinone), digoxin, vasodilators,angiotensin II converting enzyme (ACE) inhibitors (e.g. are captopril,enalapril, lisinopril, benazepril, quinapril, fosinopril, and ramipril),angiotensin II receptor blockers (ARB) (e.g. candesartan, irbesartan,olmesartan, losartan, valsartan, telmisartan, eprosartan), calciumchannel blockers, isosorbide dinitrate, hydralazine, nitrates (e.g.isosorbide mononitrate, isosorbide dinitrate), hydralazine,beta-blockers (e.g. carvedilol, metoprolol), and natriuretic peptides(e.g. nesiritide).

In certain embodiments, an additional therapy may be a pharmaceuticalagent that enhances the body's immune system, including low-dosecyclophosphamide, thymostimulin, vitamins and nutritional supplements(e.g., antioxidants, including vitamins A, C, E, beta-carotene, zinc,selenium, glutathione, coenzyme Q-10 and echinacea), and vaccines, e.g.,the immunostimulating complex (ISCOM), which comprises a vaccineformulation that combines a multimeric presentation of antigen and anadjuvant.

In certain such embodiments, the additional therapy is selected to treator ameliorate a side effect of one or more pharmaceutical compositionsof the present invention. Such side effects include, without limitation,injection site reactions, liver function test abnormalities, renalfunction abnormalities, liver toxicity, renal toxicity, central nervoussystem abnormalities, and myopathies. For example, increasedaminotransferase levels in serum may indicate liver toxicity or liverfunction abnormality. For example, increased bilirubin may indicateliver toxicity or liver function abnormality.

In certain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areadministered at the same time. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are administered at different times. Incertain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areprepared together in a single formulation. In certain embodiments, oneor more pharmaceutical compositions of the present invention and one ormore other pharmaceutical agents are prepared separately.

Certain Pharmaceutical Compositions

In certain embodiments, a compound comprising a modified oligonucleotidecomplementary to a miRNA, or precursor thereof, described herein isprepared as a pharmaceutical composition for the treatment of a disorderdescribed herein. In certain embodiments, a compound comprising amodified oligonucleotide having a nucleobase sequence complementary to amiRNA or a precursor thereof is prepared as a pharmaceutical compositionfor the prevention of a disorder described herein.

In certain embodiments, a pharmaceutical composition of the presentinvention is administered in the form of a dosage unit (e.g., tablet,capsule, bolus, etc.). In certain embodiments, such pharmaceuticalcompositions comprise a modified oligonucleotide in a dose selected from25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg,125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg,170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg,215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg,260 mg, 265 mg, 270 mg, 270 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg,305 mg, 310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg,350 mg, 355 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg,395 mg, 400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg,440 mg, 445 mg, 450 mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg,485 mg, 490 mg, 495 mg, 500 mg, 505 mg, 510 mg, 515 mg, 520 mg, 525 mg,530 mg, 535 mg, 540 mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570 mg,575 mg, 580 mg, 585 mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg, 615 mg,620 mg, 625 mg, 630 mg, 635 mg, 640 mg, 645 mg, 650 mg, 655 mg, 660 mg,665 mg, 670 mg, 675 mg, 680 mg, 685 mg, 690 mg, 695 mg, 700 mg, 705 mg,710 mg, 715 mg, 720 mg, 725 mg, 730 mg, 735 mg, 740 mg, 745 mg, 750 mg,755 mg, 760 mg, 765 mg, 770 mg, 775 mg, 780 mg, 785 mg, 790 mg, 795 mg,and 800 mg. In certain such embodiments, a pharmaceutical composition ofthe present invention comprises a dose of modified oligonucleotideselected from 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, and 800 mg.

In certain embodiments, a pharmaceutical agent is sterile lyophilizedmodified oligonucleotide that is reconstituted with a suitable diluent,e.g., sterile water for injection or sterile saline for injection. Thereconstituted product is administered as a subcutaneous injection or asan intravenous infusion after dilution into saline. The lyophilized drugproduct consists of a modified oligonucleotide which has been preparedin water for injection, or in saline for injection, adjusted to pH7.0-9.0 with acid or base during preparation, and then lyophilized. Thelyophilized modified oligonucleotide may be 25-800 mg of a modifiedoligonucleotide. It is understood that this encompasses 25, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mgof modified lyophilized oligonucleotide. The lyophilized drug productmay be packaged in a 2 mL Type I, clear glass vial (ammoniumsulfate-treated), stoppered with a bromobutyl rubber closure and sealedwith an aluminum FLIP-OFF® overseal.

In certain embodiments, the compositions of the present invention mayadditionally contain other adjunct components conventionally found inpharmaceutical compositions, at their art-established usage levels.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the oligonucleotide(s) of the formulation.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition of the presentinvention is prepared using known techniques, including, but not limitedto mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or tabletting processes.

In certain embodiments, a pharmaceutical composition of the presentinvention is a liquid (e.g., a suspension, elixir and/or solution). Incertain of such embodiments, a liquid pharmaceutical composition isprepared using ingredients known in the art, including, but not limitedto, water, glycols, oils, alcohols, flavoring agents, preservatives, andcoloring agents.

In certain embodiments, a pharmaceutical composition of the presentinvention is a solid (e.g., a powder, tablet, and/or capsule). Incertain of such embodiments, a solid pharmaceutical compositioncomprising one or more oligonucleotides is prepared using ingredientsknown in the art, including, but not limited to, starches, sugars,diluents, granulating agents, lubricants, binders, and disintegratingagents.

In certain embodiments, a pharmaceutical composition of the presentinvention is formulated as a depot preparation. Certain such depotpreparations are typically longer acting than non-depot preparations. Incertain embodiments, such preparations are administered by implantation(for example subcutaneously or intramuscularly) or by intramuscularinjection. In certain embodiments, depot preparations are prepared usingsuitable polymeric or hydrophobic materials (for example an emulsion inan acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a delivery system. Examples of delivery systemsinclude, but are not limited to, liposomes and emulsions. Certaindelivery systems are useful for preparing certain pharmaceuticalcompositions including those comprising hydrophobic compounds. Incertain embodiments, certain organic solvents such as dimethylsulfoxideare used.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises one or more tissue-specific delivery moleculesdesigned to deliver the one or more pharmaceutical agents of the presentinvention to specific tissues or cell types. For example, in certainembodiments, pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a co-solvent system. Certain of such co-solventsystems comprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a sustained-release system. A non-limiting exampleof such a sustained-release system is a semi-permeable matrix of solidhydrophobic polymers. In certain embodiments, sustained-release systemsmay, depending on their chemical nature, release pharmaceutical agentsover a period of hours, days, weeks or months.

In certain embodiments, a pharmaceutical composition of the presentinvention is prepared for oral administration. In certain of suchembodiments, a pharmaceutical composition is formulated by combining oneor more compounds comprising a modified oligonucleotide with one or morepharmaceutically acceptable carriers. Certain of such carriers enablepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a subject. In certain embodiments, pharmaceuticalcompositions for oral use are obtained by mixing oligonucleotide and oneor more solid excipient. Suitable excipients include, but are notlimited to, fillers, such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In certainembodiments, such a mixture is optionally ground and auxiliaries areoptionally added. In certain embodiments, pharmaceutical compositionsare formed to obtain tablets or dragee cores. In certain embodiments,disintegrating agents (e.g., cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate) are added.

In certain embodiments, dragee cores are provided with coatings. Incertain such embodiments, concentrated sugar solutions may be used,which may optionally contain gum arabic, talc, polyvinyl pyrrolidone,carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquersolutions, and suitable organic solvents or solvent mixtures. Dyestuffsor pigments may be added to tablets or dragee coatings.

In certain embodiments, pharmaceutical compositions for oraladministration are push-fit capsules made of gelatin. Certain of suchpush-fit capsules comprise one or more pharmaceutical agents of thepresent invention in admixture with one or more filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In certain embodiments,pharmaceutical compositions for oral administration are soft, sealedcapsules made of gelatin and a plasticizer, such as glycerol orsorbitol. In certain soft capsules, one or more pharmaceutical agents ofthe present invention are be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

In certain embodiments, pharmaceutical compositions are prepared forbuccal administration. Certain of such pharmaceutical compositions aretablets or lozenges formulated in conventional manner.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition is prepared foradministration by inhalation. Certain of such pharmaceuticalcompositions for inhalation are prepared in the form of an aerosol sprayin a pressurized pack or a nebulizer. Certain of such pharmaceuticalcompositions comprise a propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In certain embodiments using a pressurized aerosol,the dosage unit may be determined with a valve that delivers a meteredamount. In certain embodiments, capsules and cartridges for use in aninhaler or insufflator may be formulated. Certain of such formulationscomprise a powder mixture of a pharmaceutical agent of the invention anda suitable powder base such as lactose or starch.

In certain embodiments, a pharmaceutical composition is prepared forrectal administration, such as a suppositories or retention enema.Certain of such pharmaceutical compositions comprise known ingredients,such as cocoa butter and/or other glycerides.

In certain embodiments, a pharmaceutical composition is prepared fortopical administration. Certain of such pharmaceutical compositionscomprise bland moisturizing bases, such as ointments or creams.Exemplary suitable ointment bases include, but are not limited to,petrolatum, petrolatum plus volatile silicones, and lanolin and water inoil emulsions. Exemplary suitable cream bases include, but are notlimited to, cold cream and hydrophilic ointment.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a modified oligonucleotide in a therapeuticallyeffective amount. In certain embodiments, the therapeutically effectiveamount is sufficient to prevent, alleviate or ameliorate symptoms of adisease or to prolong the survival of the subject being treated.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art.

In certain embodiments, one or more modified oligonucleotides of thepresent invention is formulated as a prodrug. In certain embodiments,upon in vivo administration, a prodrug is chemically converted to thebiologically, pharmaceutically or therapeutically more active form of amodified oligonucleotide. In certain embodiments, prodrugs are usefulbecause they are easier to administer than the corresponding activeform. For example, in certain instances, a prodrug may be morebioavailable (e.g., through oral administration) than is thecorresponding active form. In certain instances, a prodrug may haveimproved solubility compared to the corresponding active form. Incertain embodiments, prodrugs are less water soluble than thecorresponding active form. In certain instances, such prodrugs possesssuperior transmittal across cell membranes, where water solubility isdetrimental to mobility. In certain embodiments, a prodrug is an ester.In certain such embodiments, the ester is metabolically hydrolyzed tocarboxylic acid upon administration. In certain instances the carboxylicacid containing compound is the corresponding active form. In certainembodiments, a prodrug comprises a short peptide (polyaminoacid) boundto an acid group. In certain of such embodiments, the peptide is cleavedupon administration to form the corresponding active form.

In certain embodiments, a prodrug is produced by modifying apharmaceutically active compound such that the active compound will beregenerated upon in vivo administration. The prodrug can be designed toalter the metabolic stability or the transport characteristics of adrug, to mask side effects or toxicity, to improve the flavor of a drugor to alter other characteristics or properties of a drug. By virtue ofknowledge of pharmacodynamic processes and drug metabolism in vivo,those of skill in this art, once a pharmaceutically active compound isknown, can design prodrugs of the compound (see, e.g., Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press, NewYork, pages 388-392).

Certain Compounds

In certain embodiments, the methods provided herein compriseadministration of a compound comprising a modified oligonucleotide. Incertain embodiments, the compound consists of a modifiedoligonucleotide.

In certain such embodiments, a compound comprises a modifiedoligonucleotide hybridized to a complementary strand, i.e. a compoundcomprises a double-stranded oligomeric compound. In certain embodiments,the hybridization of a modified oligonucleotide to a complementarystrand forms at least one blunt end. In certain such embodiments, thehybridization of a modified oligonucleotide to a complementary strandforms a blunt end at each terminus of the double-stranded oligomericcompound. In certain embodiments, a terminus of a modifiedoligonucleotide comprises one or more additional linked nucleosidesrelative to the number of linked nucleosides of the complementarystrand. In certain embodiments, the one or more additional nucleosidesare at the 5′ terminus of a modified oligonucleotide. In certainembodiments, the one or more additional nucleosides are at the 3′terminus of a modified oligonucleotide. In certain embodiments, at leastone nucleobase of a nucleoside of the one or more additional nucleosidesis complementary to the target RNA. In certain embodiments, eachnucleobase of each one or more additional nucleosides is complementaryto the target RNA. In certain embodiments, a terminus of thecomplementary strand comprises one or more additional linked nucleosidesrelative to the number of linked nucleosides of a modifiedoligonucleotide. In certain embodiments, the one or more additionallinked nucleosides are at the 3′ terminus of the complementary strand.In certain embodiments, the one or more additional linked nucleosidesare at the 5′ terminus of the complementary strand. In certainembodiments, two additional linked nucleosides are linked to a terminus.In certain embodiments, one additional nucleoside is linked to aterminus.

In certain embodiments, a compound comprises a modified oligonucleotideconjugated to one or more moieties which enhance the activity, cellulardistribution or cellular uptake of the resulting antisenseoligonucleotides. In certain such embodiments, the moiety is acholesterol moiety or a lipid moiety. Additional moieties forconjugation include carbohydrates, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. In certain embodiments, a conjugategroup is attached directly to a modified oligonucleotide. In certainembodiments, a conjugate group is attached to a modified oligonucleotideby a linking moiety selected from amino, hydroxyl, carboxylic acid,thiol, unsaturations (e.g., double or triple bonds),8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA),substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl,and substituted or unsubstituted C2-C10 alkynyl. In certain suchembodiments, a substituent group is selected from hydroxyl, amino,alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen,alkyl, aryl, alkenyl and alkynyl.

In certain such embodiments, the compound comprises a modifiedoligonucleotide having one or more stabilizing groups that are attachedto one or both termini of a modified oligonucleotide to enhanceproperties such as, for example, nuclease stability. Included instabilizing groups are cap structures. These terminal modificationsprotect a modified oligonucleotide from exonuclease degradation, and canhelp in delivery and/or localization within a cell. The cap can bepresent at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), orcan be present on both termini. Cap structures include, for example,inverted deoxy abasic caps.

Suitable cap structures include a 4′,5′-methylene nucleotide, a1-(beta-D-erythrofuranosyl) nucleotide, a 4′-thio nucleotide, acarbocyclic nucleotide, a 1,5-anhydrohexitol nucleotide, anL-nucleotide, an alpha-nucleotide, a modified base nucleotide, aphosphorodithioate linkage, a threo-pentofuranosyl nucleotide, anacyclic 3′,4′-seco nucleotide, an acyclic 3,4-dihydroxybutyl nucleotide,an acyclic 3,5-dihydroxypentyl nucleotide, a 3′-3′-inverted nucleotidemoiety, a 3′-3′-inverted abasic moiety, a 3′-2′-inverted nucleotidemoiety, a 3′-2′-inverted abasic moiety, a 1,4-butanediol phosphate, a3′-phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a3′-phosphate, a 3′-phosphorothioate, a phosphorodithioate, a bridgingmethylphosphonate moiety, and a non-bridging methylphosphonate moiety5′-amino-alkyl phosphate, a 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2-aminododecylphosphate, a hydroxypropyl phosphate, a 5′-5′-inverted nucleotidemoiety, a 5′-5′-inverted abasic moiety, a 5′-phosphoramidate, a5′-phosphorothioate, a 5′-amino, a bridging and/or non-bridging5′-phosphoramidate, a phosphorothioate, and a 5′-mercapto moiety.

Certain Nucleobase Sequences

Provided herein are methods for the treatment or prevention of adisorder such as an inflammatory disorder or a myeloproliferativedisorder. In certain embodiments, the methods comprise administration ofa pharmaceutical composition comprising a modified oligonucleotide. Incertain embodiments, the methods comprise administration of a compoundcomprising a modified oligonucleotide. In certain embodiments, amodified oligonucleotide has a sequence that is complementary to a miRNAor a precursor thereof. In certain embodiments, the miRNA is miR-155.

Nucleobase sequences of mature miRNAs and their corresponding stem-loopsequences described herein are the sequences found in miRBase, an onlinesearchable database of miRNA sequences and annotation, found athttp://microrna.sanger.ac.uk/. Entries in the miRBase Sequence databaserepresent a predicted hairpin portion of a miRNA transcript (thestem-loop), with information on the location and sequence of the maturemiRNA sequence. The miRNA stem-loop sequences in the database are notstrictly precursor miRNAs (pre-miRNAs), and may in some instancesinclude the pre-miRNA and some flanking sequence from the presumedprimary transcript. The miRNA nucleobase sequences decribed hereinencompass any version of the miRNA, including the sequences described inRelease 10.0 of the miRBase sequence database and sequences described inany earlier Release of the miRBase sequence database. A sequencedatabase release may result in the re-naming of certain miRNAs. Asequence database release may result in a variation of a mature miRNAsequence. The compounds of the present invention encompass modifiedoligonucleotides that are complementary any nucleobase sequence versionof the miRNAs described herein.

It is understood that any nucleobase sequence set forth herein isindependent of any modification to a sugar moiety, an internucleosidelinkage, or a nucleobase. It is further understood that a nucleobasesequence comprising U's also encompasses the same nucleobase sequencewherein ‘U’ is replaced by ‘T’ at one or more positions having ‘U.”Conversely, it is understood that a nucleobase sequence comprising T'salso encompasses the same nucleobase sequence wherein ‘T; is replaced by‘U’ at one or more positions having ‘T.”

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a miRNA or a precursor thereof,meaning that the nucleobase sequence of a modified oligonucleotide is aleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identicalto the complement of a miRNA or precursor thereof over a region of 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases,or that the two sequences hybridize under stringent hybridizationconditions. Accordingly, in certain embodiments the nucleobase sequenceof a modified oligonucleotide may have one or more mismatched basepairswith respect to its target miRNA or target miRNA precursor sequence, andis capable of hybridizing to its target sequence. In certainembodiments, a modified oligonucleotide has a nucleobase sequence thatis 100% complementary to a miRNA or a precursor thereof. In certainembodiments, the nucleobase sequence of a modified oligonucleotide hasfull-length complementary to a miRNA.

In certain embodiments, miR-155 has the nucleobase sequence of SEQ IDNO: 72. In certain embodiments pre-miR-155 has the nucleobase sequenceof SEQ ID NO: 72.

In certain embodiments, a modified oligonucleotide has a sequence thatis complementary to the nucleobase sequence of miR-155 set forth as SEQID NO: 73. In certain embodiments, a modified oligonucleotide has asequence that is complementary to the nucleobase sequence of pre-miR-155set forth as SEQ ID NO: 72.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence comprising the nucleobase sequence 5′-CCCCUAUCACGAUUAGCAUUAA-3′(SEQ ID NO: 74).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence consisting of the nucleobase sequence set forth as SEQ ID NO:74.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence of pre-miR-155set forth as SEQ ID NO: 72.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence having at least80% identity to the nucleobase sequence set forth in SEQ ID NO: 73. Incertain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence having at least85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least98% identity to the nucleobase sequence set forth in SEQ ID NO: 73.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence having at least80% identity to a nucleobase sequence of pre-miR-155 set forth in SEQ IDNO: 72. In certain embodiments, a modified oligonucleotide has anucleobase sequence that is complementary to a nucleobase sequencehaving at least 85%, at least 90%, at least 92%, at least 94%, at least96%, or at least 98% identity to a nucleobase sequence of pre-miR-155set forth in SEQ ID NO: 72.

In certain embodiments, a nucleobase sequence of a modifiedoligonucleotide has full-length complementary to a miRNA nucleobasesequence listed herein, or a precursor thereof. In certain embodiments,a modified oligonucleotide has a nucleobase sequence having one mismatchwith respect to the nucleobase sequence of the mature miRNA, or aprecursor thereof. In certain embodiments, a modified oligonucleotidehas a nucleobase sequence having two mismatches with respect to thenucleobase sequence of the miRNA, or a precursor thereof. In certainsuch embodiments, a modified oligonucleotide has a nucleobase sequencehaving no more than two mismatches with respect to the nucleobasesequence of the mature miRNA, or a precursor thereof. In certain suchembodiments, the mismatched nucleobases are contiguous. In certain suchembodiments, the mismatched nucleobases are not contiguous.

In certain embodiments, a modified oligonucleotide consists of a numberof linked nucleosides that is equal to the length of the mature miRNA towhich it is complementary.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is less than the length of the mature miRNA to which itis complementary. In certain such embodiments, the number of linkednucleosides of a modified oligonucleotide is one less than the length ofthe mature miRNA to which it is complementary. In certain suchembodiments, a modified oligonucleotide has one less nucleoside at the5′ terminus. In certain such embodiments, a modified oligonucleotide hasone less nucleoside at the 3′ terminus. In certain such embodiments, amodified oligonucleotide has two fewer nucleosides at the 5′ terminus.In certain such embodiments, a modified oligonucleotide has two fewernucleosides at the 3′ terminus. A modified oligonucleotide having anumber of linked nucleosides that is less than the length of the miRNA,wherein each nucleobase of a modified oligonucleotide is complementaryto each nucleobase at a corresponding position in a miRNA, is consideredto be a modified oligonucleotide having a nucleobase sequence 100%complementary to a portion of a miRNA sequence.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is greater than the length of the miRNA to which it iscomplementary. In certain such embodiments, the nucleobase of anadditional nucleoside is complementary to a nucleobase of a miRNAstem-loop sequence. In certain embodiments, the number of linkednucleosides of a modified oligonucleotide is one greater than the lengthof the miRNA to which it is complementary. In certain such embodiments,the additional nucleoside is at the 5′ terminus of a modifiedoligonucleotide. In certain such embodiments, the additional nucleosideis at the 3′ terminus of a modified oligonucleotide. In certainembodiments, the number of linked nucleosides of a modifiedoligonucleotide is two greater than the length of the miRNA to which itis complementary. In certain such embodiments, the two additionalnucleosides are at the 5′ terminus of a modified oligonucleotide. Incertain such embodiments, the two additional nucleosides are at the 3′terminus of a modified oligonucleotide. In certain such embodiments, oneadditional nucleoside is located at the 5′ terminus and one additionalnucleoside is located at the 3′ terminus of a modified oligonucleotide.

In certain embodiments, a portion of the nucleobase sequence of amodified oligonucleotide is 100% complementary to the nucleobasesequence of the miRNA, but the modified oligonucleotide is not 100%complementary over its entire length. In certain such embodiments, thenumber of nucleosides of a modified oligonucleotide having a 100%complementary portion is greater than the length of the miRNA. Forexample, a modified oligonucleotide consisting of 24 linked nucleosides,where the nucleobases of nucleosides 1 through 23 are each complementaryto a corresponding position of a miRNA that is 23 nucleobases in length,has a 23 nucleoside portion that is 100% complementary to the nucleobasesequence of the miRNA and approximately 96% overall complementarity tothe nucleobase sequence of the miRNA.

In certain embodiments, the nucleobase sequence of a modifiedoligonucleotide is 100% complementary to a portion of the nucleobasesequence of a miRNA. For example, a modified oligonucleotide consistingof 22 linked nucleosides, where the nucleobases of nucleosides 1 through22 are each complementary to a corresponding position of a miRNA that is23 nucleobases in length, is 100% complementary to a 22 nucleobaseportion of the nucleobase sequence of a miRNA. Such a modifiedoligonucleotide has approximately 96% overall complementarity to thenucleobase sequence of the entire miRNA, and has 100% complementarity toa 22 nucleobase portion of the miRNA.

In certain embodiments, a portion of the nucleobase sequence of amodified oligonucleotide is 100% complementary to a portion of thenucleobase sequence of a miRNA, or a precursor thereof. In certain suchembodiments, 15 contiguous nucleobases of a modified oligonucleotide areeach complementary to 15 contiguous nucleobases of a miRNA, or aprecursor thereof. In certain such embodiments, 16 contiguousnucleobases of a modified oligonucleotide are each complementary to 16contiguous nucleobases of a miRNA, or a precursor thereof. In certainsuch embodiments, 17 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 17 contiguous nucleobases of amiRNA, or a precursor thereof. In certain such embodiments, 18contiguous nucleobases of a modified oligonucleotide are eachcomplementary to 18 contiguous nucleobases of a miRNA, or a precursorthereof. In certain such embodiments, 19 contiguous nucleobases of amodified oligonucleotide are each complementary to 19 contiguousnucleobases of a miRNA, or a precursor thereof. In certain suchembodiments, 20 contiguous nucleobases of a modified oligonucleotide areeach complementary to 20 contiguous nucleobases of a miRNA, or aprecursor thereof. In certain such embodiments, 22 contiguousnucleobases of a modified oligonucleotide are each complementary to 22contiguous nucleobases of a miRNA, or a precursor thereof. In certainsuch embodiments, 23 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 23 contiguous nucleobases of amiRNA, or a precursor thereof. In certain such embodiments, 24contiguous nucleobases of a modified oligonucleotide are eachcomplementary to 24 contiguous nucleobases of a miRNA, or a precursorthereof.

Certain Modified Oligonucleotides

In certain embodiments, a modified oligonucleotide consists of 12 to 30linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 15 to 25 linked nucleosides. In certain embodiments, amodified oligonucleotide consists of 19 to 24 linked nucleosides. Incertain embodiments, a modified oligonucleotide consists of 21 to 24linked nucleosides.

In certain embodiments, a modified oligonucleotide consists of 12 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 13 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 14 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 15 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 16 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 17 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 18 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 19 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 20 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 21 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 22 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 23 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 24 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 25 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 26 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 27 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 28 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 29 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 30 linkednucleosides. In certain such embodiments, a modified oligonucleotidecomprises linked nucleosides selected from contiguous nucleobases of SEQID NO: 74.

Certain Modifications

Modified oligonucleotides of the present invention comprise one or moremodifications to a nucleobase, sugar, and/or internucleoside linkage. Amodified nucleobase, sugar, and/or internucleoside linkage may beselected over an unmodified form because of desirable properties suchas, for example, enhanced cellular uptake, enhanced affinity for otheroligonucleotides or nucleic acid targets and increased stability in thepresence of nucleases.

In certain embodiments, a modified oligonucleotide of the presentinvention comprises one or more modified nucleosides. In certain suchembodiments, a modified nucleoside is a stabilizing nucleoside. Anexample of a stabilizing nucleoside is a sugar-modified nucleoside.

In certain embodiments, a modified nucleoside is a sugar-modifiednucleoside. In certain such embodiments, the sugar-modified nucleosidescan further comprise a natural or modified heterocyclic base moietyand/or a natural or modified internucleoside linkage and may includefurther modifications independent from the sugar modification. Incertain embodiments, a sugar modified nucleoside is a 2′-modifiednucleoside, wherein the sugar ring is modified at the 2′ carbon fromnatural ribose or 2′-deoxy-ribose.

In certain embodiments, a 2′-modified nucleoside has a bicyclic sugarmoiety. In certain such embodiments, the bicyclic sugar moiety is a Dsugar in the alpha configuration. In certain such embodiments, thebicyclic sugar moiety is a D sugar in the beta configuration. In certainsuch embodiments, the bicyclic sugar moiety is an L sugar in the alphaconfiguration. In certain such embodiments, the bicyclic sugar moiety isan L sugar in the beta configuration.

In certain embodiments, the bicyclic sugar moiety comprises a bridgegroup between the 2′ and the 4′-carbon atoms. In certain suchembodiments, the bridge group comprises from 1 to 8 linked biradicalgroups. In certain embodiments, the bicyclic sugar moiety comprises from1 to 4 linked biradical groups. In certain embodiments, the bicyclicsugar moiety comprises 2 or 3 linked biradical groups. In certainembodiments, the bicyclic sugar moiety comprises 2 linked biradicalgroups. In certain embodiments, a linked biradical group is selectedfrom O, S, N(R₁)—, —C(R₁)(R₂)—, —C(R₁)═C(R₁)—, —C(R₁)═N—, —C(═NR₁)—,—Si(R₁)(R₂)—, —S(═O)₂—, —S(═O)⁻, —C(═O)— and —C(═S)—; where each R₁ andR₂ is, independently, H, hydroxyl, C1-C12 alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, aheterocycle radical, a substituted heterocycle radical, heteroaryl,substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇alicyclic radical, halogen, substituted oxy (—O—), amino, substitutedamino, azido, carboxyl, substituted carboxyl, acyl, substituted acyl,CN, thiol, substituted thiol, sulfonyl (S(═O)₂—H), substituted sulfonyl,sulfoxyl (S(═O)—H) or substituted sulfoxyl; and each substituent groupis, independently, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, amino, substituted amino, acyl, substituted acyl, C₁-C₁₂aminoalkyl, C₁-C₁₂ aminoalkoxy, substituted C₁-C₁₂ aminoalkyl,substituted C₁-C₁₂ aminoalkoxy or a protecting group.

In some embodiments, the bicyclic sugar moiety is bridged between the 2′and 4′ carbon atoms with a biradical group selected from —O—(CH₂)_(p)—,—O—CH₂—, —O—CH₂CH₂—, —O—CH(alkyl)-, —NH—(CH₂)_(p)—,—N(alkyl)-(CH₂)_(p)—, —O—CH(alkyl)-, —(CH(alkyl))-(CH₂)_(p)—,—NH—O—(CH₂)_(p)—, —N(alkyl)-O—(CH₂)_(p)—, or —O—N(alkyl)-(CH₂)_(p)—,wherein p is 1, 2, 3, 4 or 5 and each alkyl group can be furthersubstituted. In certain embodiments, p is 1, 2 or 3.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from halo, allyl, amino, azido, SH, CN,OCN, CF₃, OCF₃, O—, S—, or N(R_(m))-alkyl; O—, S—, or N(R_(m))-alkenyl;O—, S— or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl,aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n))or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl. These 2′-substituent groups can be furthersubstituted with one or more substituent groups independently selectedfrom hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂),thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), —O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, OCF₃, O—CH₃, OCH₂CH₂OCH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, andO—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, O—CH₃, and OCH₂CH₂OCH₃.

In certain embodiments, a sugar-modified nucleoside is a 4′-thiomodified nucleoside. In certain embodiments, a sugar-modified nucleosideis a 4′-thio-2′-modified nucleoside. A 4′-thio modified nucleoside has aβ-D-ribonucleoside where the 4′-O replaced with 4′-S. A4′-thio-2′-modified nucleoside is a 4′-thio modified nucleoside havingthe 2′-OH replaced with a 2′-substituent group. Suitable 2′-substituentgroups include 2′-OCH₃, 2′-O—(CH₂)₂—OCH₃, and 2′-F.

In certain embodiments, a modified oligonucleotide of the presentinvention comprises one or more internucleoside modifications. Incertain such embodiments, each internucleoside linkage of a modifiedoligonucleotide is a modified internucleoside linkage. In certainembodiments, a modified internucleoside linkage comprises a phosphorusatom.

In certain embodiments, a modified oligonucleotide of the presentinvention comprises at least one phosphorothioate internucleosidelinkage. In certain embodiments, each internucleoside linkage of amodified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a modified internucleoside linkage does notcomprise a phosphorus atom. In certain such embodiments, aninternucleoside linkage is formed by a short chain alkyl internucleosidelinkage. In certain such embodiments, an internucleoside linkage isformed by a cycloalkyl internucleoside linkages. In certain suchembodiments, an internucleoside linkage is formed by a mixed heteroatomand alkyl internucleoside linkage. In certain such embodiments, aninternucleoside linkage is formed by a mixed heteroatom and cycloalkylinternucleoside linkages. In certain such embodiments, aninternucleoside linkage is formed by one or more short chainheteroatomic internucleoside linkages. In certain such embodiments, aninternucleoside linkage is formed by one or more heterocyclicinternucleoside linkages. In certain such embodiments, aninternucleoside linkage has an amide backbone. In certain suchembodiments, an internucleoside linkage has mixed N, O, S and CH₂component parts.

In certain embodiments, a modified oligonucleotide comprises one or moremodified nucleobases. In certain embodiments, a modified oligonucleotidecomprises one or more 5-methylcytosines. In certain embodiments, eachcytosine of a modified oligonucleotide comprises a 5-methylcytosine.

In certain embodiments, a modified nucleobase is selected from5-hydroxymethyl cytosine, 7-deazaguanine and 7-deazaadenine. In certainembodiments, a modified nucleobase is selected from 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. In certainembodiments, a modified nucleobase is selected from 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, a modified nucleobase comprises a polycyclicheterocycle. In certain embodiments, a modified nucleobase comprises atricyclic heterocycle. In certain embodiments, a modified nucleobasecomprises a phenoxazine derivative. In certain embodiments, thephenoxazine can be further modified to form a nucleobase known in theart as a G-clamp.

Certain Oligonucleotide Motifs

Suitable motifs for modified oligonucleotides of the present inventioninclude, but are not limited to, fully modified, uniformly modified,positionally modified, and gapmer. Modified oligonucleotides having afully modified motif, including a uniformly modified motif, may bedesigned to target mature miRNAs. Alternatively, modifiedoligonucleotides having a fully modified motif, including a uniformlymodified motif, may be designed to target certain sites of pri-miRNAs orpre-miRNAs, to block the processing of miRNA precursors into maturemiRNAs. Modified oligonucleotides having a fully modified motif oruniformly modified motif are effective inhibitors of miRNA activity.

In certain embodiments, a fully modified oligonucleotide comprises asugar modification at each nucleoside. In certain such embodiments,pluralities of nucleosides are 2′-O-methoxyethyl nucleosides and theremaining nucleosides are 2′-fluoro nucleosides. In certain suchembodiments, each of a plurality of nucleosides is a 2′-O-methoxyethylnucleoside and each of a plurality of nucleosides is a bicyclicnucleoside. In certain such embodiments, a fully modifiedoligonucleotide further comprises at least one modified internucleosidelinkage. In certain such embodiments, each internucleoside linkage of afully sugar-modified oligonucleotide is a modified internucleosidelinkage. In certain embodiments, a fully sugar-modified oligonucleotidefurther comprises at least one phosphorothioate internucleoside linkage.In certain such embodiments, each internucleoside linkage of a fullysugar-modified oligonucleotide is a phosphorothioate internucleosidelinkage.

In certain embodiments, a fully modified oligonucleotide is modified ateach internucleoside linkage. In certain such embodiments, eachinternucleoside linkage of a fully modified oligonucleotide is aphosphorothioate internucleoside linkage.

In certain embodiments, a uniformly modified oligonucleotide comprisesthe same sugar modification at each nucleoside. In certain suchembodiments, each nucleoside of a modified oligonucleotide comprises a2′-O-methoxyethyl sugar modification. In certain embodiments, eachnucleoside of a modified oligonucleotide comprises a 2′-O-methyl sugarmodification. In certain embodiments, each nucleoside of a modifiedoligonucleotide comprises a 2′-fluoro sugar modification. In certainsuch embodiments, a uniformly modified oligonucleotide further comprisesat least one modified internucleoside linkage. In certain suchembodiments, each internucleoside linkage of a uniformly sugar-modifiedoligonucleotide is a modified internucleoside linkage. In certainembodiments, a uniformly sugar-modified oligonucleotide furthercomprises at least one phosphorothioate internucleoside linkage. Incertain such embodiments, each internucleoside linkage of a uniformlysugar-modified oligonucleotide is a phosphorothioate internucleosidelinkage.

In certain embodiments, a uniformly modified oligonucleotide comprisesthe same internucleoside linkage modifications throughout. In certainsuch embodiments, each internucleoside linkage of a uniformly modifiedoligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide comprises the samesugar modification at each nucleoside, and further comprises one or moreinternucleoside linkage modifications. In certain such embodiments, themodified oligonucleotide comprises one modified internucleoside linkageat the 5′ terminus and one modified internucleoside linkage at the 3′terminus. In certain embodiments, the modified oligonucleotide comprisestwo modified internucleoside linkages at the 5′ terminus and twomodified internucleoside linkages at the 3′ terminus. In certainembodiments, the modified oligonucleotide comprises two modifiedinternucleoside linkages at the 5′ terminus and three modifiedinternucleoside linkages at the 3′ terminus. In certain embodiments, themodified oligonucleotide comprises two modified internucleoside linkagesat the 5′ terminus and four modified internucleoside linkages at the 3′terminus. In certain such embodiments, the modified internucleosidelinkage is a phosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide is represented by thefollowing formula III:

(S′)QxQz¹(Qy)_(n)Qz₂Qz³Qz⁴Q-L(3′)

In certain such embodiments, an compound is represented by formula III.In certain embodiments, Q is a 2′-O-methyl modified nucleoside. Incertain embodiments, x is phosphorothioate. In certain embodiments, y isphosphodiester. In certain embodiments, each of z1, z2, z3, and z4 is,independently phosphorothioate or phosphodiester. In certainembodiments, n is 6 to 17. In certain embodiments, L is cholesterol. Incertain embodiments, n is 12 to 17.

In certain embodiments, x is

One of A and B is S while the other is O;

-   -   y is

Each of z1, z2, z3, and z4 is independently x or y;

-   -   n=6-17    -   L is

Wherein:

-   -   X is N(CO)R⁷, or NR′;    -   Each of R¹, R³ and R⁹, is independently, H, OH, or —CH₂OR^(b)        provided that at least one of R¹, R³ and R⁹ is OH and at least        one of R¹, R³ and R⁹ is —CH₂OR^(b);    -   R⁷ is R^(d) or C₁-C₂₀ alkyl substituted with NR^(c)R^(d) or        NHC(O)R^(d);    -   R^(c) is H or C₁-C₆ alkyl;    -   R^(d) is a carbohydrate radical; or a steroid radical, which is        optionally tethered to at least one carbohydrate radical; and    -   R^(b) is

with one of A and B is S while the other is O.

In certain embodiments, R^(d) is cholesterol. In certain embodimentseach of z¹, z², z³, and z⁴ is

with one of A and B is S while the other is O.

In certain embodiments, R¹ is —CH₂OR^(b). In certain embodiments, R⁹ isOH. In certain embodiments, R¹ and R⁹ are trans. In certain embodiments,R⁹ is OH. In certain embodiments, R¹ and R³ are trans. In certainembodiments, R3 is —CH₂OR^(b). In certain embodiments, R¹ is OH. Incertain embodiments, R¹ and R³ are trans. In certain embodiments, R⁹ isOH. In certain embodiments, R³ and R⁹ are trans. In certain embodiments,R⁹ is CH₂OR^(b). In certain embodiments, R¹ is OH. In certainembodiments, R¹ and R⁹ are trans. In certain embodiments, X is NC(O)R⁷.In certain embodiments, R⁷ is —CH₂(CH₂)₃CH₂NHC(O)R^(d).

A modified oligonucleotide having a gapmer motif may have an internalregion consisting of linked 2′-deoxynucleotides, and external regionsconsisting of linked 2′-modified nucleosides. Such a gapmer may bedesigned to elicit RNase H cleavage of a miRNA precursor. The internal2′-deoxynucleoside region serves as a substrate for RNase H, allowingthe cleavage of the miRNA precursor to which a modified oligonucleotideis targeted. In certain embodiments, each nucleoside of each externalregion comprises the same 2′-modified nucleoside. In certainembodiments, one external region is uniformly comprised of a first2′-modified nucleoside and the other external region is uniformlycomprised of a second 2′-modified nucleoside.

A modified oligonucleotide having a gapmer motif may have a sugarmodification at each nucleoside. In certain embodiments, the internalregion is uniformly comprised of a first 2′-modified nucleoside and eachof the wings is uniformly comprised of a second 2′-modified nucleoside.In certain such embodiments, the internal region is uniformly comprisedof 2′-fluoro nucleosides and each external region is uniformly comprisedof 2′-O-methoxyethyl nucleosides.

In certain embodiments, each external region of a gapmer consists oflinked 2′-O-methoxyethyl nucleosides. In certain embodiments, eachexternal region of a gapmer consists of linked 2′-O-methyl nucleosides.In certain embodiments, each external region of a gapmer consists of2′-fluoro nucleosides. In certain embodiments, each external region of agapmer consists of linked bicyclic nucleosides.

In certain embodiments, each nucleoside of one external region of agapmer comprises 2′-O-methoxyethyl nucleosides and each nucleoside ofthe other external region comprises a different 2′-modification. Incertain such embodiments, each nucleoside of one external region of agapmer comprises 2′-O-methoxyethyl nucleosides and each nucleoside ofthe other external region comprises 2′-O-methyl nucleosides. In certainsuch embodiments, each nucleoside of one external region of a gapmercomprises 2′-O-methoxyethyl nucleosides and each nucleoside of the otherexternal region comprises 2′-fluoro nucleosides. In certain suchembodiments, each nucleoside of one external region of a gapmercomprises 2′-O-methyl nucleosides and each nucleoside of the otherexternal region comprises 2′-fluoro nucleosides. In certain suchembodiments, each nucleoside of one external region of a gapmercomprises 2′-O-methoxyethyl nucleosides and each nucleoside of the otherexternal region comprises bicyclic nucleosides. In certain suchembodiments, each nucleoside of one external region of a gapmercomprises 2′-O-methyl nucleosides and each nucleoside of the otherexternal region comprises bicyclic nucleosides.

In certain embodiments, nucleosides of one external region comprise twoor more sugar modifications. In certain embodiments, nucleosides of eachexternal region comprise two or more sugar modifications. In certainembodiments, at least one nucleoside of an external region comprises a2′-O-methoxyethyl sugar and at least one nucleoside of the same externalregion comprises a 2′-fluoro sugar. In certain embodiments, at least onenucleoside of an external region comprises a 2′-O-methoxyethyl sugar andat least one nucleoside of the same external region comprises a bicyclicsugar moiety. In certain embodiments, at least one nucleoside of anexternal region comprises a 2′-O-methyl sugar and at least onenucleoside of the same external region comprises a bicyclic sugarmoiety. In certain embodiments at least one nucleoside of an externalregion comprises a 2′-O-methyl sugar and at least one nucleoside of thesame external region comprises a 2′-fluoro sugar. In certainembodiments, at least one nucleoside of an external region comprises a2′-fluoro sugar and at least one nucleoside of the same external regioncomprises a bicyclic sugar moiety.

In certain embodiments, each external region of a gapmer consists of thesame number of linked nucleosides. In certain embodiments, one externalregion of a gapmer consists a number of linked nucleosides differentthat that of the other external region.

In certain embodiments, the external regions comprise, independently,from 1 to 6 nucleosides. In certain embodiments, an external regioncomprises 1 nucleoside. In certain embodiments, an external regioncomprises 2 nucleosides. In certain embodiments, an external regioncomprises 3 nucleosides. In certain embodiments, an external regioncomprises 4 nucleosides. In certain embodiments, an external regioncomprises 5 nucleosides. In certain embodiments, an external regioncomprises 6 nucleosides. In certain embodiments, the internal regionconsists of 17 to 28 linked nucleosides. In certain embodiments, aninternal region consists of 17 to 21 linked nucleosides. In certainembodiments, an internal region consists of 17 linked nucleosides. Incertain embodiments, an internal region consists of 18 linkednucleosides. In certain embodiments, an internal region consists of 19linked nucleosides. In certain embodiments, an internal region consistsof 20 linked nucleosides. In certain embodiments, an internal regionconsists of 21 linked nucleosides. In certain embodiments, an internalregion consists of 22 linked nucleosides. In certain embodiments, aninternal region consists of 23 linked nucleosides. In certainembodiments, an internal region consists of 24 linked nucleosides. Incertain embodiments, an internal region consists of 25 linkednucleosides. In certain embodiments, an internal region consists of 26linked nucleosides. In certain embodiments, an internal region consistsof 27 linked nucleosides. In certain embodiments, an internal regionconsists of 28 linked nucleosides.

Certain Quantitation Assays

The effects of antisense inhibition of a miRNA following theadministration of modified oligonucleotides may be assessed by a varietyof methods known in the art. In certain embodiments, these methods arebe used to quantitate miRNA levels in cells or tissues in vitro or invivo. In certain embodiments, changes in miRNA levels are measured bymicroarray analysis. In certain embodiments, changes in miRNA levels aremeasured by one of several commercially available PCR assays, such asthe TaqMan® MicroRNA Assay (Applied Biosystems). In certain embodiments,antisense inhibition of a miRNA is assessed by measuring the mRNA and/orprotein level of a target of a miRNA. Antisense inhibition of a miRNAgenerally results in the increase in the level of mRNA and/or protein ofa target of the miRNA.

Certain Experimental Models

In certain embodiments, the present invention provides methods of usingand/or testing modified oligonucleotides of the present invention in anexperimental model. In certain embodiments, experimental models areemployed to evaluate the effectiveness of modified oligonucleotides ofthe invention for the treatment of inflammation and/ormyeloproliferative disorders. Those having skill in the art are able toselect and modify the protocols for such experimental models to evaluatea pharmaceutical agent of the invention.

Modified oligonucleotides may be tested in primary cells or culturedcells. Suitable cell types include those that are related to the celltype to which delivery of a modified oligonucleotide is desired in vivo.For example, suitable cell types for the study of miR-155 antagonists,including modified oligonucleotides, include macrophages, monocytes,granulocytes, lymphocytes, neutrophils, hematopoietic stem cells, andmyeloid cells.

In some embodiments, candidate compounds are tested for their activityas miR-155 antagonists in an in vivo model. For example, in someembodiments, a candidate compound can be administered to a mouse, e.g.,a mouse that has been irradiated and reconstituted with hematopoieticstem cells (HSC's) that express miR-155 as described, for example, inExample 9 herein. Following administration of a candidate compound tothe reconstituted mice, the bone marrow, thymus, spleen and lymph nodescan be removed and assessed for histological changes associated withmiR-155 expression in HSC's, such as myeloproliferative disorderspresent in the bone marrow, splenomegaly, evidence of extramedullaryhematopoiesis, and perturbation of peripheral blood cell populations, asdescribed herein. In some embodiments, bone marrow, splenocytes, andperipheral blood cells of the mice can be analyzed for cell surfacemarkers as a means to assess the presence and amount of certain celltypes in bone marrow, spleens, peripheral blood, etc. of the mice. Forexample, bone marrow, splenocytes, and peripheral blood can be analyzedby fluorescence activated cell sorting (FACS) for the presence andamount of Mac1⁺, CD4⁺, B220⁺, Ter-199⁺, and Gr1⁺-expressing cells, andthe like, as described herein. Levels of miR-155 expression in bonemarrow, splenocytes, peripheral blood cells obtained from the micetreated with the candidate compound can be measured. The histology oforgans and tissues, the cell population/distribution, and the geneexpression measurements in the mice treated with the candidate compoundcan be compared to untreated mice (e.g., mice reconstituted with miR155expressing HSC's) as well as control mice (e.g., non-irradiated,non-reconstituted). A decrease in the expression of miR-155 in certaincell populations, a decrease in the extent of splenomegaly or otherhistological changes associated with miR-155 expression, or decrease inthe cell population changes associated with miR-155 expression comparedto reconstituted mice that did not receive the candidate compoundindicates that the candidate compound can be used therapeutically as anmiR-155 antagonist.

In certain embodiments, the extent to which a modified oligonucleotideinhibits the activity of a miRNA is assessed in primary cells orcultured cells. In certain embodiments, inhibition of miRNA activity maybe assessed by measuring the levels of the miRNA. Alternatively, thelevel of a predicted or validated miRNA target may be measured. Aninhibition of miRNA activity may result in the increase in the mRNAand/or protein of a miRNA target. Further, in certain embodiments,certain phenotypic outcomes may be measured. For example, suitablephenotypic outcomes include inhibition of cell proliferation, theinduction of cell death, and/or the induction of apoptosis.

Following the in vitro identification of a modified oligonucleotide thateffectively inhibits the activity of a miRNA, modified oligonucleotidesare further tested in in vivo experimental models, such as thosedescribed in the Examples.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention.

EXAMPLES Example 1—IFN-β and Poly (I:C) Induce Expression of miR-155 inMacrophages

The following example describes experiments to test whether virallyrelevant stimuli induce expression of miRNA's.

Macrophages were matured from murine bone marrow and stimulated witheither the synthetic viral intermediate poly (I:C) (double-strandedRNA), or the host antiviral response cytokine, IFN-β. To obtain themacrophages, bone marrow cells were isolated from the tibias and femursof mice as described in Doyle et al. (2002) Immunity 17:251-263. For theexperiments described herein, WT, MyD88^(−/−), TRIF^(−/−), IFNAR^(−/−),and TNFR1^(−/−) mice, all of which are on a C57BL/6 genetic background,were bred and housed in the University of California Division ofLaboratory Animal Medicine facility and killed according to establishedprotocols approved by the Animal Research Committee. Bone marrow wascollected using routine protocols, and RBCs were lysed by using a RBClysis buffer (Invitrogen, San Diego, Calif.). The remaining bone marrowcells were plated out in DMEM containing 10% FBS, 100 units/mlpenicillin, and 100 units/ml streptomycin and supplemented withmacrophage colony-stimulating factor-conditioned medium at a previouslyestablished concentration. Cells were cultured in a humidified incubatorwith 5% CO₂ at 37° C. After 7 days of culture, a portion of themacrophages were stained with specific antibodies and analyzed by FACSto ensure proper differentiation (CD11b⁺F4/80⁺CD11c⁻) and subsequentlyused for experiments.

For FACS, RBC-depleted splenocytes were stained in FACS buffer (1×PBS/0.1% BSA/2% FBS/0.1% normal mouse serum) by usingphycoerythrin-conjugated anti-CD11b or FITC-conjugated anti-CD86(eBiosciences, San Diego, Calif.) and fixed with paraformaldehyde (1%final concentration). Surface expression was assayed by using a FACScan®flow cytometer (Becton Dickenson, Franklin Lakes, N.J.).

Primary macrophages were stimulated by using fresh DMEM containing oneof the following: 2 μg/ml poly(I:C) (Amersham Pharmacia, Piscataway,N.J., 1,000 units/ml mIFN-β (R&D Systems, Minneapolis, Minn.). The cellswere stimulated for six hours, at which time total RNA was isolated fromthe cells and used in microarray screening, quantitative PCR (qPCR), orNorthern Blotting.

The microarray screening procedure is the same as described in Taganov,et al. (2006) Proc. Nat. Acad. Sci. USA 103:12481-12486. RNA fromstimulated macrophages was collected by using the mirVana® RNA isolationkit (Ambion, Austin, Tex.); 30 μg was enriched for small RNAs, tailed byusing the mirVana® miRNA labeling kit (Ambion), and labeled with eitherCy3 (control samples) or Cy5 (stimulated samples) fluorescent dyes(Amersham Pharmacia, Piscataway, N.J.). The stimulated and controlsamples were next mixed and incubated for 14 h with miRNA array slides.The epoxy-coated slides (Schott-Nexterion, Louisville, Ky.) wereprepared in quadruplicate by using robotics for the spotting of 200mouse and human sequences complimentary to different mammalian miRNAs(mirVana® miRNA Probe Set; Ambion, Austin, Tex.). After hybridization,microarrays were scanned with a GenePix®c 4200A scanner (AxonInstruments, Foster City, Calif.) by using Gene Pix 5.0 software (AxonInstruments). Raw data were imported into the Resolver gene expressiondata analysis system version 4.0 (Rosetta Biosoftware, Seattle, Wash.)for further processing. The microarray data for IFN-β stimulated cellsare shown in FIG. 1A. The microarray data for poly (I:C) stimulatedcells are shown in FIG. 1B. miR-155 was the only miRNA substantiallyinduced by both poly (I:C) and IFN-β. FIGS. 1A and 1B.

To confirm the microarray data showing that miR-155 transcript wasinduced by IFN-β and poly (I:C), the total RNA isolated as describedabove was used in quantitative PCR (qPCR) and Northern Blot analysis.For qPCR, total RNA was harvested from bone-marrow-derived macrophagesby using the TRIzol® Reagent (Invitrogen) according to themanufacturer's protocol. 1 μg of total RNA was converted to cDNA byusing iScript® (Invitrogen, Carlsbad, Calif.) according to themanufacturer's protocol. Cybergreen-based real-time qPCR was performedby using the 7300 Real-Time PCR system (Applied Biosystems, Foster City,Calif.) and gene-specific primers for TNF-α, IP10, and L32 as describedin Doyle, et al. (2002) Immunity 17:251-263; Kracht et al., (2002)Cytokine 20:91-106. All qPCR data were normalized to L32 values. Todetect the expression of L32 mRNA, cDNA was subjected to PCR and run outon a 2% agarose gel containing ethidium bromide at 1 μg/ml. The primersequences used to detect BIC were 5′-ttggcctctgactgactcct-3′(forward)(SEQ ID NO: 1) and 5′-gcagggtgactcttggactt-3′ (reverse) (SEQ IDNO: 2). The relative levels of expression of miR-155 in culturesstimulated with culture medium alone (m), INF-β, or poly (I:C) weredetermined. The data are shown in FIG. 2A. qPCR confirmed induction ofmiR-155 expression in macrophages in response to stimulation with IFN-βand poly (I:C). qPCR also confirmed that the small nuclear RNA U6 is notinduced in macrophages by either IFN-β or poly (I:C) stimulation (FIG.3A). As expected, IP10 is an IFN-β target. IP10 mRNA was induced by bothIFN-β and poly (I:C) (FIG. 3B).

For detection of miR-155 by Northern blotting, RNA was extracted byusing the TRIzol® RNA extraction reagent (Invitrogen, Carlsbad, Calif.)according to the manufacturer's protocol. 15 μg of total RNA waselectrophoretically separated on a 12% polyacrylamide denaturing gel,and tRNA was visualized by using ethidium bromide staining to ensure thequality and relative amount of the RNA. (FIG. 2C). Total RNA was nexttransferred to a GeneScreenPlus membrane (PerkinElmer, Boston, Mass.) byusing a semidry Transblot electrophoresis apparatus (Bio-Rad, Hercules,Calif.). The RNA was crosslinked to the membrane by using UV radiation.Hybridization was carried out by using ULTRAHybOligo® solution (Ambion,Austin, Tex.) according to the manufacturer's instructions. The probesequence was complementary to the mature form of miR-155, and waslabeled with γ-³²P. After washing, the membranes were imaged by using aSTORM® phosphorimager (GE Healthcare Life Sciences, Piscataway, N.J.).Detection of miR-155 and U6 was also performed by using the mirVana®qRT-PCR miRNA detection kit (Ambion, Austin, Tex.) according to themanufacturer's instructions. FIG. 2B is an image of the Northern Blot.The Northern Blot confirmed the induced expression of miR-155 inmacrophages stimulated with either IFN-β or poly (I:C).

To determine whether the macrophage marker CD11b and the IFN-β targetgene were induced after 24 hours of stimulation with either IFN-β orpoly (I:C), RBC-depleted splenocytes were stained in FACS buffer(1×PBS/0.1% BSA/2% FBS/0.1% normal mouse serum) by usingphycoerythrin-conjugated anti-CD11b or FITC-conjugated anti-CD86(eBiosciences, San Diego, Calif.) and fixed with paraformaldehyde (1%final concentration). Surface expression was assayed by using a FACScan®flow cytometer (Becton Dickenson, Franklin Lakes, N.J.). The FACS dataare presented in FIGS. 4A-4C. Expression of the cell surface marker CD86was up-regulated following stimulation with both poly (I:C) (FIG. 4B)and IFN-β (FIG. 4D).

The data presented in this example indicate that macrophages respond toviral cues by strongly up-regulating miR-155, an miRNA that is knownfrom other studies to function as an oncogene.

Example 2—Kinetics of miR-155 Induction in Macrophages Following Poly(I:C) and IFN-β Stimulation

miR-155 is found within the BIC gene on chromosome 21 in humans and 16in mice. The genomic structure of human BIC consists of three exons, andits transcript is transcribed and processed into two differently sizedmRNA molecules through alternative polyadenylation. BIC lacks a largeORF and therefore is unlikely to encode a protein. Without intending tobe bound by any particular theory, it is possible that the sole functionof BIC may be to give rise to miR-155 encoded within exon 3. An unscaledgraphical depiction of the genomic structure of the human BIC noncodingRNA gene is shown in FIG. 5A. The location of miR-155 within exon 3 isshown (155). The sequence of human BIC cDNA (SEQ ID NO: 71), pre-miR-155(SEQ ID NO: 72) and mature miR-155 (SEQ ID NO: 73) from homo sapiens areknown. Eis et al. (2005) Proc. Nat. Acad. Sci. USA 102:3627-3632.

To monitor the kinetics of miR-155 induction, both BIC mRNA and maturemiR-155 were assayed over a 48-h time course after poly(I:C) or IFN-βstimulation of primary macrophages. Briefly, murine macrophages wereisolated and stimulated with culture medium (medium), poly (I:C), orIFN-β over 48 hours as described in Example 1. Samples of the cultureswere harvested for RNA isolation at 0 hours, 8 hours, 24 hours, and 48hours post-stimulation. Total RNA was isolated as described inExample 1. Reverse transcription with an oligonucleotiide dT primer wasperformed as described in Example 1. The cDNA was used as a template inPCR and run out on a 2% agarose gel containing ethidium bromide at 1μg/ml. The primer sequences used to detect BIC were5′-ttggcctctgactgactcct-3′ (forward) (SEQ ID NO: 1) and5′-gcagggtgactcttggactt-3′ (reverse) (SEQ ID NO: 2). The relative levelsof expression of miR-155 in cultures stimulated with culture mediumalone (m), INF-β, or poly (I:C) were determined. Detection of L32 wasdetermined as described in Example 1. The data are shown in FIG. 5B.

A portion of the RNA was used in qPCR to detect miR-155 (mature) overthe same 48 hour time period. qPCR was performed as described inExample 1. The relative expression of miR-155 mRNA is shown on alogarithmic scale at the indicated time points, in FIG. 5C. In responseto poly(I:C), BIC mRNA became detectable by 2 h, remained elevated to 8h, and was still present at reduced levels by 24 and 48 h afterstimulation. FIG. 5B. miR-155 induction by poly(I:C) followed a similarpattern of expression as BIC, with the exception of remaining at itshighest levels at the 24- and 48-h time points. FIG. 5C. IFN-β did notinduce BIC mRNA by 2 h, but it was detected by 8 h and was nearlyundetectable by 24 and 48 h. FIG. 5B. IFN-β induction of miR-155followed the same delayed pattern of induction as BIC, reaching itshighest levels by 8 h and slowly decreasing by 24 and 48 h afterstimulation. FIG. 5C.

These findings demonstrate that the regulation of miR-155 levelsinvolves BIC mRNA up-regulation by poly(I:C) or IFN-β Furthermore, thesedata show that miR-155 is an immediate early target gene ofpoly(I:C)-induced signaling, whereas its induction is relatively delayeddownstream from IFN-β stimulation.

Example 3—Various Toll-Like Receptor (TLR) Ligands Induce miR-155 inMacrophages

TLR3 is a receptor for poly(I:C). The following experiments wereconducted to determine whether other TLR ligands induce miR-155.Specifically, the experiments described below assess the affects ofstimulation with LPS, which signals through TLR4; hypomethylated DNA(CpG), a TLR9 ligand; and Pam3CSK4, a synthetic lipoprotein thatactivates TLR2, on miR-155 expression in primary macrophages.

Macrophages were isolated from murine bone marrow as described inExample 1. The macrophages were stimulated by using fresh DMEMcontaining one of the following: 2 μg/ml poly(I:C) (Amersham Pharmacia,Piscataway, N.J.); 5 ng/ml 055-B5 LPS (Sigma, St. Louis, Mo.), 100 nMCpG 1668 oligonucleotides (Invitrogen, Carlsbad, Calif.), 2 μg/mlPam3CSK4 (Invitrogen, Carlsbad, Calif.). After 6 h, RNA was harvestedfrom these cells and analyzed by Northern Blot to detect BIC mRNA, ormature miR-155, as described in Example 1. The data are shown in FIG. 6.These data demonstrate that all four TLR ligands tested strongly inducedmiR-155 expression, and show that miR-155 is induced by the same TLR'sthat recognize pathogen-associated molecular patterns from viruses andother pathogens.

Example 4—Toll-Like Receptor Adaptors are Required for miR-155 Inductionin Response to TLR Ligands in Macrophages

TLRs signal through the MyD88 family of adaptor proteins. Of theseadaptors, TLR2 and TLR9 signaling is known to require MyD88, whereasTLR3 utilizes TRIF. Adaptors can also play partially redundant roles;for instance, TLR4 signals through either MyD88 or TRIF. This exampledescribes experiments to determine whether MyD88 and TRIF adaptors arerequired for TLR induction of miR-155.

Briefly, macrophages were isolated from WT mice, or mice deficient ineither TRIF or MyD88 as described in Example 1. The isolated macrophageswere stimulated for 6 hours with either medium (m), the TLR ligands poly(I:C) (p:(I:C)), LPS, CpG or Pam3CSK4 (P3C), as described in Example 1.Total RNA was isolated from the cells, and relative levels of miR-155expression were measured by qPCR as described in Example 1. The data arepresented in FIG. 7A. CpG (TLR9)- or Pam3CSK4 (TLR2)-treated macrophagesrequired MyD88, but not TRW, to induce miR-155. FIG. 7A. On the otherhand, poly(I:C) (TLR3) required TRIF but not MyD88. TLR4 up-regulatedmiR-155 in the absence of either single adaptor.

These data confirm the specificity of the TLR ligands used anddemonstrate that either MyD88- or TRIF-dependent signaling pathways aresufficient to induce miR-155.

A subset of TLR-responsive genes require IFN-β autocrine/paracrinesignaling for their induction. Because miR-155 is up-regulated by bothTLRs and IFN-β the following experiments were conducted to determinewhether TLR induction of miR-155 required IFN-β autocrine/paracrinesignaling. Macrophages were isolated from both wild-type (WT) andIFNAR^(−/−) mice, as described in Example 1. Macrophages were stimulatedwith poly(I:C), LPS, CpG, or Pam3CSK4 as described above. miR-155expression was measured by qPCR as described in Example 1. The data arepresented in FIG. 7B. TLRs do not require IFN-β production for earlyup-regulation of miR-155 in response to stimulation with poly (I:C),LPS, CpG, or Pam3CSK4. FIG. 7B.

Example 5—IFN's Induce miR-155 Expression Through TNF-αAutocrine/Paracrine Signaling

Similar to IFN-α, IFN-γ is produced in response to viral and bacterialinfections and plays an important role in macrophage activation.Accordingly, the following experiments were carried out to determinewhether IFN-γ also induced miR-155 expression in macrophages. BecauseIFN induction of BIC mRNA and mature miR-155 was delayed compared withthat of poly(I:C), it appeared that IFNs might use a proteinintermediate to up-regulate miR-155. TNF-α has been shown to haveautocrine/paracrine signaling after IFN-γ stimulation of macrophages.Accordingly, experiments set forth below were conducted to determinewhether IFN induction of miR-155 required TNF-α autocrine/paracrinesignaling.

Briefly, macrophages were isolated from wild-type (WT) and TNFR1^(−/−)mice as described in Example 1. The macrophages were stimulated withmedium (m), 1,000 units/ml IFN-β, 50 ng/ml IFN-γ, or 10 ng/ml TNF-α for6 h, as described in Example 1. miR-155 was assayed by qPCR as describedin Examples 1-3. The data are presented in FIGS. 8A and 8B. IFN-γinduced miR-155 in macrophages after 6 hours stimulation. FIG. 8A. Thiseffect was dependent upon TNFR. FIG. 8A. IFN-β and IFN-γ failed toup-regulate miR-1′55 in the absence of TNFR1 signaling as compared withthe induction in WT cells. FIG. 8A. TNF-α was sufficient to inducemiR-155 expression in a TNFR1-dependent manner. FIG. 8B.

To test whether IFN-β and IFN-γ induced TNF-α expression, WT macrophageswere stimulated with culture medium (m), IFN-β, or IFN-γ for 6 h, andTNF-α mRNA levels were determined by qPCR as described in Examples 1-3.The Same RNA was also used in qPCR to measure the amount of IP10expression following stimulation with culture medium or IFN-β, asdescribed in Example 1. The results are shown in FIGS. 8C and 8D. BothIFN-β and IFN-γ induced TNF-α expression (FIG. 8C). IFN-β induction ofIP10 remained intact in TNFR1^(−/−) macrophages (FIG. 8D). These datademonstrate that TNFR1^(−/−) cells can still respond to IFN treatment.

Next, WT macrophages were stimulated with medium or 2 μg/ml poly(I:C)for 6 h and assayed for TNF-α expression by qPCR as described above. Inaddition, WT and TNFR1^(−/−) macrophages were stimulated with medium orpoly(I:C) for 6 h, and miR-155 was assayed by qPCR. Poly (I:C) inducedTNF-α expression. The poly (I:C) induction of miR-155 did not requireTNF-α auto-signaling (FIGS. 8E, 8F).

Together, these data demonstrate that TNF-α is an inducer of miR-155 andthat IFN's require TNF-α. Finally, whereas poly (I:C) induced TNF-αexpression, this induction did not require TNF-α autorcine/paracrinesignaling to up-regulate miR-155 in macrophages.

Example 6—The JNK Pathway is Involved in Up-Regulation of miR-155 InResponse to Poly (I:C) and TNF-α

The following experiments were conducted to identify signaling pathwaysinvolved in miR-155 induction. Sequence analysis of the promoter regionof the BIC gene conserved between mice and humans, showed two putativeAP-1 transcription factor binding sites. The AP-1 transcriptionalcomplex is known to be activated by inflammatory stimuli including TLRligands and TNF-α. The following experiments were conducted to testwhether the JNK pathway is involved in imR-155 induction.

WT murine macrophages were isolated as described in Example 1 andtreated for 30 minutes with either 5 μm/mL SP600125, 25 μm/mL SP600125,uo126 (Calbiochem, La Jolla, Calif.) dissolved in DMSO, or DMSO alone.The macrophages were subsequently stimulated with culture medium (m),poly (I:C), or TNF-α, as described in Example 1, for 4 hours. miR-155expression was measured by qPCR as described in Example 1. The data arepresented in FIGS. 9A-9B and 10. Vehicle-treated cells up-regulatedmiR-155 levels by 4 hours after stimulation, whereas the JNK inhibitorSP600125 blocked miR-155 induction by both poly (I:C) and TNF-α in adose-dependent manner. FIGS. 9A-9B, 10. As a control, the ERK inhibitoruo126 did not reduce poly (I:C) induction of miR-155 (data not shown).

These data indicate that the JNK pathway is involved in theup-regulation of miR-155 expression in response to poly (I:C) or TNF-α.

Example 7—LPS Induces Bone Marrow Expression of miR155 In Vivo

To confirm the in vitro effects observed in Examples 1-6, the followingexperiments were conducted to determine whether miR-155 expression iselevated in the bone marrow compartment after the onset of inflammationin vivo.

In the experiments described in the Examples below, wild-type (WT)C57Bl6 mice purchased from The Jackson Laboratory were used. Rag1^(−/−)mice were bred in house. All experiments involved female mice and wereperformed according to IACUC-approved protocols.

WT mice were injected i.p. with either 50 μg LPS (Sigma-Aldrich) inphosphate buffered saline (PBS), or with PBS alone. Bone marrow cellswere harvested at 24 hours or 72 hours post-treatment as described inExample 1. RNA was isolated from total bone marrow cells as described inExample 1, and analyzed for miR-155 expression using qPCR as describedin Example 1. The data are shown in FIG. 11A. miR-155 was stronglyinduced in bone marrow cells 24 hours after LPS treatment, and returnedto control levels by 72 hours post-treatment.

To determine whether up-regulation of miR-155 expression in isolatedbone marrow cells by LPS or GM-CSF stimulation was attributable to cellsother than mature B and T lymphocytes, miR-155 expression in bone marrowcells isolated from either WT or Rag1^(−/−) mice was measured, followingstimulation with culture medium, LPS, or GM-CSF. WT and Rag1^(−/−) micewere injected with 100 ng/ml LPS (Sigma Aldrich), 100 ng/ml GM-CSF (GM)(eBioscience), or culture medium (M), as described in Example 1. Bonemarrow was flushed out of the femurs and tibias of WT and Rag1^(−/−)mice (n=3 per group) 24 hours after stimulation, and RNA was isolatedfrom the total bone marrow cells as described in Example 1. qPCR wasperformed to determine the levels of miR-155 expression as described inExample 1. The data are presented in FIG. 11B. NC corresponds to notemplate control for the qPCR reaction. Up-regulation of miR-155expression was detected upon LPS and GM-CSF stimulation in bone marrowcells from both WT and Rag1^(−/−) mice, demonstrating that cells otherthan mature B and T lymphocytes contribute to this response.

Example 8—LPS Induces Expansion of GM Cells, Reduction in B Cells, andReduction in Erythroid Precursors

The following experiments were conducted to determine whether LPSaffects bone marrow cell dynamics in vivo.

WT mice were injected i.p. with either PBS or 50 μg/mL LPS dissolved inPBS. 72 hours post-injection, bone marrow cells were isolated from themice. Fluorophor-conjugated monoclonal antibodies specific for Mac1,Gr1, Ter-199, B220 and CD4 (eBioscience, San Diego, Calif.) were used invarious combinations to stain bone marrow cells that were fixed afterwashing using paraformaldehyde (1% final). Stained cells were assayedusing a BD FACSCalibur® flow cytometer (Becton Dickenson, FranklinLakes, N.J.) and further analyzed with FloJo® FACS analysis software(Becton Disckenson, Franklin Lakes, N.J.). The FACS data are presentedin FIGS. 12A-12F. The percentage of GM cells (expressing Mac1 and Gr1surface markers) was substantially increased 72 hours post-injectionwith LPS, as compared to PBS-injected control mice. FIGS. 12A-12B. Bycontrast, there was a substantial reduction in the percentage of B cells(expressing the cell surface marker B220) 72 hours post-injection withLPS, compared to PBS-injected control mice. FIGS. 12C-12D. Similarly,there was a susbtantial reduction in the percentage of erythroidprecursor cells (expressing the cell surface marker Ter-119) 72 hourspost-injection with LPS, compared to PBS-injected control mice. FIGS.12E-12F.

Bone marrow cells isolated from WT mice injected with PBS or 50m/mL LPSdissolved in PBS 72 hours post-injection, as described above, werestained with Wright's stain (Wright) or hematoxylin and eosin (H&E)stains using routine protocols. The results are shown in FIGS. 13A-13D.The micro-photographs of bone marrow from LPS-treated mice showedmyeloid preponderance and hyperplasia, with relative erythroidhyperplasia.

These data, along with the data showing maximal induction of miR-155expression at 24 hours post-LPS treatment (Example 7), indicate thatLPS-induced miR-155 expression in the bone marrow precedes GM cellexpansion.

Example 9—Forced Expression of miR-155 in HSC's CausesMyeloproliferative Disorder in Bone Marrow

The following experiments were conducted to determine whether miR-155 issufficient to mediate GM expansion in vivo in mouse bone marrow.

To generate vectors for expression miR-155, an miR-155 expressioncassette containing the human miR-155 hairpin sequence and flankingregions was cloned from a cDNA library into pcDNA3 as described in Eis,P. S., et al. (2005) Proc. Natl. Acad. Sci. USA 102:3627-3632, hereinexpressly incorporated by reference in its entirety. The cassette wassubcloned into pMSCVpuro (Clonetech, Mountainview, Calif.), FUW (Lois etal., (2002) Science 295:868-872), or pMG (Invivogen, San Diego, Calif.),using routine molecular cloning techniques. pMG155 is a modified pMSCVvector whereby GFP was placed downstream from the 5′LTR, and the miR-155expression cassette was cloned downstream from the GFP stop codon usingroutine cloning techniques. FIG. 14A is graphical depiction (not toscale) of the retroviral construct used to enable both mir-155 and GFPexpression in HSCs.

HSC-enriched bone marrow cells were obtained by injecting mice i.p. with5 μg 5-fluorouracil for 5 days before bone marrow harvest, as describedin Yang et al. (2005) Proc. Nat. Acad. Sci. USA 102:4518-4523, thecontents of which is herein expressly incorporated by reference in itsentirety. Cells were collected from the bone marrow, and RBCs wereremoved using an RBC lysis solution (Invitrogen, Carlsbad, Calif.) asdescribed in Example 1. Cells were cultured for 24 h. in 20 ng/ml IL-3,50 ng/ml IL-6, and 50 ng/ml SCF (all from eBioscience, San Diego,Calif.) containing complete RPMI (10% FBS, 100 U/ml penicillin, 100 U/mlstreptomycin and 50 μM β-mercaptoethanol) before initial retroviralinfection. To generate retroviurses for infecting HSC-enriched bonemarrow cells, 293T cells were transfected with pMG155 and pCL-Eco(Imgenex, San Diego, Calif.) using a standard calcium phosphateprotocol. After 48 h, 8 μg/ml polybrene was added to culturesupernatant-containing retroviruses. The culture supernatant was used tospin-infect 10⁶ HSC-enriched cells per donor for 1.5 h at 2,500 RPM and30° C. This procedure was repeated three times once daily, followed byinjection of 10⁶ retrovirally infected HSC-enriched cells per lethallyirradiated (1,100 rads from Cesium 137 source at 50 rads/minute)recipient mice. Recipient mice were maintained on Septra throughout thereconstitution period.

Two months after reconstitution, mice were sacrificed. Bone marrow,thymus, spleen and lymph nodes were analyzed for GFP and miR-155co-expression. Bone marrow was isolated as described in Example 1. Aportion of the bone marrow was analyzed by FACs, as described in Example1, to assess GFP-expression. The FACS data are presented in FIGS. 14Band 14C. The percentage of GFP bone marrow cells of mice reconstitutedwith control vector (Cont, FIG. 14B) and MG155 vector (MG155, FIG. 14C)is indicated. Total RNA was isolated from whole bone marrow and used togenerate cDNA for quantitative PCR as described in Example 1. miR-155RNA levels were measured as described in Example 1. The data arepresented in FIG. 14D. The data show that control mice, or mice injectedwith the GFP control vector did not show detectable levels of miR-155.By contrast, miR-155 expression was detected in cells isolated from micereconstituted with HSC's transfected with the MG155 vector.

Tibias and femurs were removed from mice reconstituted with MG155 orcontrol vector HSCs for two months, or untreated C57BL6 control (B6)mice. FIG. 15 is a photograph of an exemplary tibia from each group ofmice. Mice expressing miR-155 exhibited white-tan bone marrowcoloration, whereas the control mice exhibited vibrant red bone marrowcoloration.

Bone marrow smears were prepared from extracted bone marrow of micereconstituted with HSC's transfected with the control vector or theMG155 vector. The preparation was air-dried and stained with Hematoxylin& Eosin (H&E) (FIGS. 16A-16B) or Wright's stain (FIGS. 16C-16D). Thepreparations were examined on an Olympus BX-511 microscope andphotographed using a Spot Digital Camera and software. The morphology ofthe bone marrow cells revealed that miR-155 expressing bone marrow wasdominated by GM cells at a variety of either normal or abnormaldevelopmental stages. Bone marrow of mice reconstituted with miR-155expressing HSC's also exhibited a reduction in erythrocytes,megakaryocytes, and lymphocytes. FIGS. 17A-17E are photographs showingan enlarged view of examples of dysplastic myeloid cells in miR-155expressing bone marrow (enlargement of indicated sections of FIG. 16C).Many of the cells that appeared to be granulocytic precursors showedirregular segmentation of the nuclei and lacked condensation of nuclearchromatin.

A portion of the bone marrow isolated from mice reconstituted with HSC'stransfected with the control vector or MG155 vector was depleted ofRBC's as described in Example 1. The cells were treated withfluorophor-conjugated Mac1, Gr1, Ter-119, B220 and CD4 monoclonalantibodies as described in Example 1. The cell preparations wereanalyzed by FACS, as described in Example 1. The number of Mac1/Gr1,Ter-119, CD4, and B220 expressing bone marrow cells is graphicallyrepresented in FIG. 18. The bone marrow of MG155-reconstituted micecontained approximately twice as many Mac1/Gr1-expressing cells, veryfew Ter-119+ erythroid precursors, and a reduction in B220+ B cells ascompared to the control vector.

GFP⁺-gated bone marrow cells from mice reconstituted with MG155 orcontrol vector HSCs were analyzed for FSC and SSC counts and expressionof Mac1 and Gr1. FIGS. 19A-19D are plots of the FACS data. GFP⁺ cellsexpressing miR-155 showed a dramatic increase in large granular cells,as defined by having high forward scatter (FSC) and side scatter (SSC)(FIGS. 19A-19B). Back-gating was used to confirm that these cells wereMac1⁺, with a majority also positive for Gr1 (FIGS. 19C-19D). FIGS.20A-20F are plots of FACS of the bone marrow. The cells responsible forthe overall GM, B, and erythroid precursor differences between MG155 andcontrol vector cells were largely GFP+.

The data above demonstrate that expression of miR-155 in bone marrowcells leads to profound myeloid proliferation with dysplastic changes,when compared with controls.

Example 10—Forced Expression of miR-155 in HSC's Causes Splenomegaly andExtramedullary Hematopoiesis

The following experiments were conducted to assess the effect of forcedmiR-155 expression in HSC's on the morphology and cellular constitutionof spleens.

Generation of HSC's stably transformed with either a GFP (control) ormiR-155/GFP expression (MG155) vector is described in Example 9.Reconstitution of lethally-irradiated mice with transformed HSC's isdescribed in Example 9.

Two months after reconstitution, spleens were removed from the mice andphotographed. Splenomegaly was observed in the mice reconstituted withmiR-155-expressing HSC's. Mice reconstituted with HSC's transformed withcontrol vector were normal. FIG. 21.

Spleens were placed into 10% neutral-buffered formalin immediately afternecropsy, fixed for 12-18 hours, washed and transferred to 70% ethanol,and embedded in paraffin using standard protocols. Spleens weresectioned and stained with hematoxylin & eosin (H&E) (FIGS. 22A-22B) orWright's stain (FIGS. 22C-22D). H&E staining revealed expandedinterfollicular regions containing various hematopoietic elements, aswell as constricted and disrupted B cell follicles compared with controlspleens. Wright's staining revealed a large number of erythroidprecursors, megakaryocytes, and some developing GM cells in the spleensof mice reconstituted with miR-155 expressing HSC's, whereas very few ofthese cell types were observed in control spleens.

Splenocytes were also analyzed by FACS to determine cell type andexpression of cell surface markers, as described in Example 9. Thenumbers of Mac1/Gr1, Ter-119, CD4 and B220 expressing splenocytes frommice reconstituted with HSC's expressing GFP alone (control), or HSC'sexpressing MG155 are indicated in FIG. 23. As expected, the spleens frommice reconstituted with MG155 HSC's had elevated numbers of Mac1⁺/Gr1⁺myeloid cells and Ter-119⁺ erythroid cells, with little change in CD4⁺ Tcells and B220⁺ B cells.

GFP⁺-gated splenocytes from mice reconstituted with MG155 or controlvector HSCs were analyzed for FSC and SSC counts and expression of Mac1and Gr1. FIGS. 24A-24D are plots of the FACS data. GFP⁺ cells expressingmiR-155 showed a dramatic increase in large granular cells, as definedby having high forward scatter (FSC) and side scatter (SSC) (FIGS.24A-24B). Back-gating was used to confirm that these cells were Mac1⁺,with a majority also positive for Gr1 (FIGS. 24C-24D). FIGS. 25A-25H areplots of FACS of the splenocytes, determining the co-distribution ofcells expressing Mac1 and GFP (FIGS. 25A-215B), Ter-119 and GFP (FIGS.25C-25D), CD4 and GFP (FIGS. 25E-25F) and B220 and GFP (FIGS. 25G-25H).miR-155-expressing splenocytes contained overall higher numbers of Mac1⁺cells, that expressed GFP compared with controls. Conversely, theTer-119⁺ cell population of miR-155 expressing spleens was largelynegative for GFP, possibly arising from non-transduced HSC's.

The data above demonstrate the presence of splenic extramedullaryhematopoiesis in miR-155 expressing mice, likely compensating for thebone marrow defects in these mice.

Example 11—Forced Expression of miR-155 in HSC's Perturbs PeripheralBlood Cell Populations

The following experiments were conducted to assess the effect of forcedmiR-155 expression in HSC's on the cellular consitution of peripheralblood.

Generation of HSC's stably transformed with either a GFP (control) ormiR-155/GFP expression (MG155) vector is described in Example 10.Reconstitution of lethally-irradiated mice with transformed HSC's isdescribed in Example 10.

Peripheral blood was collected from mice reconstituted with MG155 orcontrol vector HSC's for two months and analyzed by FACS to correlateGFP expression and FSC and SSC counts, as well as to determine theexpression of Mac1 as described in Example 1. The data are presented inFIGS. 26A-26D. The total number of Mac1⁺ cells was also determined (FIG.26E). By two months after reconstitution, there were significantlyelevated numbers of Mac1⁺ cells in the peripheral blood of micereconstituted with MG155, compared to mice reconstituted with controlvector HSC's.

Peripheral blood smears were prepared from the mice reconstituted withHSC's transfected with the control vector or the MG155 vector. Thepreparation was air-dried and stained with Wright's stain (FIGS.27A-16D). The preparations were examined on an Olympus BX-511 microscopeand photographed using a Spot Digital Camera and software. FIGS. 27A-27Care photographs of Wright-stained monocytes and neutrophils,respectively, from mice reconstituted with HSC's transfected withcontrol vector. FIGS. 27C-27D are photographs of exemplaryWright-stained myeloid cells from mice reconstituted with HSC'stransfected with MG155. The morphology of the peripheral blood cells ofmiR-155 expressing mice was abnormal. FIGS. 29A-29B are photographs ofWright-stained RBC's from mice reconstituted with HSC's transfected withthe control vector (FIG. 29A), or HSC's transfected with the miR-155expression vector (FIG. 29B). The photographs show several immatureerythrocytes demonstrating polychormatophilia in miR-155 expressinganimals. Data represent at least nine independent animals in each group,and p-values (*) of <0.05 were considered significant after a Student'stwo-tailed test.

Complete blood cell counts were performed on the blood of reconstitutedmice. The data are presented in FIGS. 28A-28F. The cell counts revealeda significant reduction in red blood cell, hemoglobin and plateletlevels in mice reconstituted with MG155-expressing HSC's compared tomice reconstituted with control vector HSC's.

Example 12—Human AML Patients Have Increased miR-155 Expression

Several of the pathological features observed in miR-155 expressing miceare associated with human myeloid malignancies, including acute myeloidleukemia (AML). The following experiments were performed to assessmiR-155 expression levels in the bone marrow of human AML patientscompared to healthy patients.

Bone marrow samples from 24 AML patients and 6 healthy subjects werecollected and flash frozen at −80° C. The samples were rapidly thawed.Total RNA was isolated from the bone marrow samples using a TRIzol® RNApurification kit (Invitrogen, Carlsbad, Calif.) according to themanufacturer's instructions. AML cases were categorized according to theWHO “Classification of Tumors” using anonymous clinical reports. The RNAwas used in quantitative PCR (qPCR) to determine miR-155 and 5Sexpression levels as described in Example 1. FIGS. 29A-29B show thelevels of miR-155 and 5S expression, respectively, in bone marrow fromnormal subjects and AML patients. Overall, on average, the AML samplessignificantly overexpressed miR-155 compared with healthy donors, with alevel approximately 4.5 times higher. A few AML samples had miR-155levels that were lower than the normal samples, whereas the overall AMLsample distribution had a wide variance. In contrast, no significantdifference in the average expression levels of 5S RNA was observedbetween the groups. FIGS. 29C-29D show the levels of miR-155 and 5Sexpression, respectively, in bone marrow from normal subjects, subjectswith M4 AML, and subjects with M5 AML. Patients with acutemyelomonocytic leukemia and acute monocytic leukemia, corresponding toFAB-AML-M4 and FAB-AML-M5, respectively, exhibited significantoverepxession of miR-155 compared with normal samples.

The data above demonstrate that miR-155 expression in the bone marrow issignificantly elevated in a subset of patients suffering from AML.

Example 13—miR-155 Directly Represses Genes Implicated in HematopoieticDevelopment and Disease

The following experiments were conducted to identify miR-155 targetgenes that may be involved in the myeloproliferative phenotype observedin the experiments described above.

miR-155 expressing retroviruses were created as described in Example 9.RAW 264.7 cells were transduced with the MSCVpuro-155 or emptyexpression vector, or empty control vector, as described in Example 9.Briefly, to generate VsVg-pseudotyped retroviruses containing themiR-155 expression cassette, 2×10⁶ 293 T cells were transfected withpMSCVpuro-miR-155, pGag-Pol and pVsVg using a standard calcium phosphateprotocol. After 48 hours, viral supernatant was harvested and used toinfect 5×10⁵ RAW 264.7 cells for 8 h in the presence of polybrene at 10μg/ml. After 48 h, stably transduced cells were selected using puromycinat 7 μg/ml for 7-10 days.

Total RNA was isolated using the RNeasy® mini RNA preparation kit(Qiagen, Valencia, Calif.), according to the manufacturer'sinstructions. The RNA was labeled and used to probe the Affymetrix MouseGenome 430 2.0 microarray according to the protocols provided by themanufacturer (available at the websiteaffymetrix.com/products/arrays/sepcific/mouse430_2. affx). Microarraydata were analyzed using the Rosetta Resolver software and deposited inthe GEO database under accession number GSE10467.

1,080 transcripts were down-regulated >1.2-fold with a p-value of <0.08.89 of the repressed mRNA's contained conserved (human and mouse) miR-155binding sites with 7- or 8-mer seeds in their 3/UTR's, according topublished lists of computationally predicted target genes found on theTargetscan 4.0 website (See, Grimson, et al. (2007) Mol. Cell 27:91-105;see also, Lewis et al. (2003) Cell 115:787-798, the entire contents ofwhich are hereby expressly incorporated by reference in theirentireties). Genes with reported roles in myeloid hyperplasia and/orhematopoiesis were identified through literature searching. Thefollowing genes were selected for further analysis, based on themicroarry data, the presence of miR-155 binding sites, and theliterature search: Bach1, Sla, Cutl1, Csf1r, Jarid2, Cebpβ, PU.1, Arntl,Hif1α, and Picalm. To confirm the microarray results, quantitative PCRwas performed on the RNA from the transduced cells using thegene-specific primers listed in Table A.

TABLE A PRIMER SEQUENCES USED FOR QUANTITATIVE PCR Primer Name SequenceSEQ ID NO: Bach1 F TGAGTGAGAGTGCGGTATTTGC  3 Bach1 RGTCAGTCTGGCCTACGATTCT  4 Sla F ATGGGGAATAGCATGAAATCAC  5 Sla RGGAGATGGGTAGTCAGTCAGC  6 Cut11 F CGCAGAGAACTGTTCATTGAGG  7 Cut11 RGAGCTGAAGGTGAGTCGCT  8 Csf1r F TGTCATGCAGCCTAGTGGC  9 Csf1r RCGGGAGATTCAGGGTCCAAG 10 Jarid2 F GAAGGCGGTAAATGGGCTTCT 11 Jarid2 RTCGTTGCTAGTAGAGGACACTT 12 Cebpβ F GACAAGCACAGCGACGAGTA 13 Cebpβ RAGCTGCTCCACCTTCTTCTG 14 PU.1 F ATGTTACAGGCGTGCAAAATGG 15 PU.1 RTGATCGCTATGGCTTTCTCCA 16 Arnt1 F ACCACAGGAACTTCTAGGTACAT 17 Arnt1 RGGACATTGGCTAAAACAACAGTG 18 Hif1a F ACCTTCATCGGAAACTCCAAAG 19 Hif1a RACTGTTAGGCTCAGGTGAACT 20 Picalm F GTCTGTCCACGCCATGTCG 21 Picalm RTAGCAGAGAAAGGATCTCCCC 22

The microarray data and qPCR data are shown in FIG. 30. qPCR values werenormalized to L32 mRNA levels and displayed as percent expression ofcontrol. Data are the average of three independent experiments. Bach1,Sla, Cutl1, Csf1r, Jarid2, Cebpβ, PU.1, Arntl, Hif1α, and Picalm weredown-regulated approximately 20%-70% in RAW 264.7 cells expressingmiR-155 versus empty vector control. Western blotting was performedusing standard protocols and the following antibodies from Santa CruzBiotechnology: Cebpβ (C-19), PU.1 (T-21), Cutl1 (M-222), Picalm (C-18),and α Tubulin (AA12). Protein expression differences were determinedusing Scion Image Software. Western Blot confirmed repression of proteinlevels of Cepbβ, PU.1, Cutl1, and Picalm, in cells expressing miR-155.FIG. 31A is an image of an exemplary Western Blot.

To determine whether miR-155 could directly repress the identified mRNAtargets through 3′UTR interactions, full length 3′UTRs, or in the caseof Bach1 and Cebpβ the region of the UTR containing the miR-155 bindingsites was cloned into pmiReport® (Ambion, Austin, Tex.), afteramplification from a mouse macrophage cDNA library. Primer sequences forthe cloning are listed in Table B. The Bach1 3′ UTR region was amplifiedfrom a human B cell library. The 2-mer control insert consists of atandem repeat of the complementary sequence to the mature mouse miR-155sequences. Cloning of the TRAF6 and IRAK1 3″ UTRs into pmiReport® wasperformed as described in Taganov, et al. (2006) Proc. Nat. Acad. Sci.USA 103:1241-12486, the contents of which are herein incorporated byreference in their entirety. 8×10⁴ 293 T cells were plated in DMEM mediacontaining 5% FBS for 18 hours, followed by transfection of relevantplasmids using lipofectamine (Invitrogen, Carlsbad, Calif.), permanufacturer's instructions. Luciferase assays were preformed 48 hoursafter transfection using a dual luciferase kit (Promega, Madison, Wis.)according to the manufacturer's protocols. A β-galactosidase expressionvector was co-transfected with the luciferase reporter constructs.β-galactosidase levels were assayed and sued to normalize the luciferasevalues. The data are shown in FIG. 31. Luciferase expression of theindicated constructs was repressed between 25-78%. TA rough correlationbetween the quantitative PCR results in RAW 264.7 and luciferaserepression in 293T cells was observed.

TABLE B PRIMERS USED FOR REPORTER CONSTRUCT CLONING Primer SEQ ID NameSEQUENCE NO: Bach1 F CCAGAGCTTAAATATAATTTGTAAAGC 23 Bach1 RACATTGAGAAGGCCAGTTCATAA 24 Sla F GTAACTAGTTGACCTGGCTTGTACACACAC 25 Sla RGTTAAGCTTTAAATACATGATTTGGCAAAGTGTAA 26 Cut11 FTCAAGAGCTCGGCAAAATCGCCATAGGC 27 Cut11 RAGCTACGCGTCCCTTCCTAACAATCAGATTAATAAAAT 28 Csf1r FGTAACTAGTTCCTGCCGCTCTCTACGT 29 Csf1r R GTTAGCTTCTGGCTGTGTTAATGCTGTTAGTT30 Jarid2 F GTAACTAGTAGATGCCGAACCCATGGT 31 Jarid2 RGTTAAGCTTATGAAGAGAAAAAATAGACAAGAGGA 32 Cebpf3 FGTAACTAGTTGCAATCCGGATCAAACG 33 Cebpf3 R GTTAAGCTTGGCTTTTAAACATTCTCCAAAAA34 PU.1 F GTAACTAGTCCGGCCATAGCATTAACC 35 PU.1 RGTTAAGCTTGGGAGAATGACTGTCAATAATTTTACT 36 Arnt1 FGTAACTAGTACACTACATTTGCTTTGGCAAC 37 Arnt1 RAGCTACGCGTAGAACAAGGGAAACATTTATTAAAAAT 38 Hif1a FTCAAGAGCTCCTGAGCGTTTCCTAATCTCATTC 39 Hif1a RAGCTACGCGTCCTGGTCCACAGAAGATGTTT 40 Picalm FTCAAGAGCTCATGGAAGAGAATGGAATTACTCCA 41 Picalm RGTTAAGCTTTGTTTTGTGGAAGCTGCATT 42

To demonstrate a direct interaction between miR-155 and the 3″ UTR'stested, site-directed mutagenesis of the reporter vectors was used tochange specific nucleotides found within the miR-155 seed regions. See,Table C. The 2-mer control miR-155 construct was repressed approximately80%. However, luciferase levels were relatively unaffected when theTraf6 or Irak1 3′ UTR's were tested, consistent with their lack ofmiR-155 binding sites.

TABLE C MIR-155 WT AND MUTANT SEED SEQUENCES miR-155 SEQ ID SEQ ID  SeedWT NO: Mutant NO: Bach1 1 AGCATTAA 43 AGGTAAAA 57 Bach1 2 AGCATTA 44AGGTAAA 58 PU.1 AGCATTAA 45 AGGTAAAA 59 Cut11 1 AGCATTAA 46 AGGTAAAA 60Cut11 2 GCATTA 47 GCTAAA 61 Picalm 1 GCATTAA 48 GGAGTGA 62 Picalm 2AGCATTA 49 AGCTAAA 63 Arnt1 1 GCATTAA 50 GCAATA 64 Arnt1 2 GCATTAA 51AGCAAATA 65 Csf1r AGCATTAA 52 AGCAAAT 66 Sla AGCATTA 53 GCAAATA 67Jarid2 1 AGCATTAA 54 ACGTAATA 68 Jarid2 2 AGCATTAA 55 AGCAAATA 69 Hif1aAGCATTA 56 AGCAAAT 70

The data presented above demonstrate that miR-155 can directly regulateseveral genes, including but not limited to Bach1, PU.1, Cutl1, Picalm,Arntl, Csf1r, Sla, Jarid2, and HIf1a, that are relevant to hematopoiesisand myeloproliferation.

Example 14—Diagnosis of Myeloproliferative Disorders

This example illustrates the diagnosis of a subject having, or suspectedof having a myeloproliferative disorder. A subject suffering from, or atrisk of developing a myeloproliferative disorder is identified. Forexample, a subject that exhibits symptoms of acute myeloid leukemia, orthat has been identified as being at risk of developing acute myeloidleukemia is identified. For example, an individual exposed to a riskfactor(s) associated with AML, e.g., carcinogens including but notlimited to benzene, tobacco smoke, and ionizing radiation, or who hasreceived chemotherapy to treat other cancers, or who has been diagnosedas having myelodysplasia, is identified. The subject can also exhibitone or more symptoms including fatigue, fever, recurrent infections,weight loss, night sweats, or bleeding.

A bone marrow sample is obtained from the subject. RNA is isolated fromthe bone marrow. The level of miR-155 (either mature or pre-miR-155) ismeasured, for example, by qPCR (as described in Example 12), or bymicroarray analysis (as described in Example 1, modified for a humanarray). Other methods known to those skilled in the art can be used todetect miR-155 levels. The miR-155 levels from the bone marrow of thesubject are compared to miR-155 levels in a control. The subject isidentified as having AML if the subject's miR-155 levels aresignificantly elevated (e.g., 2-fold, 3-fold, 4-fold, 5-fold, or more,or any number in between) compared to miR-155 levels in normal subjects.

Example 15—Treatment of Myeloproliferative Disorders

This example illustrates the treatment of a subject suffering from amyeloproliferative disorder, such as acute myeloid leukemia (AML).

A subject suffering from or at risk of developing a myeloidproliferative disease or disorder is identified. The subject isidentified by any means known to those skilled in the art, including themethods described in Example 14 herein. The subject is administered aneffective amount of an miR-155 antagonist, such as an miR-155 antisensecompound. A typical daily dose for an miR-155 antagonist might rangefrom about 0.01 μg/kg to about 1 mg/kg of patient body weight or moreper day, depending on the factors mentioned above. Preferably the doseranges from about 10 μg/kg/day to about 100 μg/kg/day. The appropriatedosage and treatment regimen can be readily determined by the skilledartisan based on a number of factors, including but not limited to thenature of the miR-155 antagonist, the route of administration, and thesubject's disease state. AML treatment efficacy is evaluated byobserving a delay or slowing of the disease progression, amelioration orpalliation of the disease state or symptoms, and/or remission.

Example 16—Treatment or Modulation of Inflammation

This example illustrates the treatment of a subject suffering frominflammation, or inflammatory-related conditions, such as inflammationarising from a macrophage-induced inflammatory response, mediatedthrough a Toll-like Receptor(s) (TLRs). The inflammation can arise as aresult of activation of TLR2, TLR3, TLR4, TLR9, pathways, or the like,for example caused by cancer, viral infection, microbial infection orthe like, as described herein.

A subject suffering from or at risk of developing a condition associatedwith TLR-mediated inflammation is identified. The subject isadministered an effective amount of an miR-155 antagonist, such as anmiR-155 antisense compound. A typical daily dose for an miR-155antagonist might range from about 0.01 μg/kg to about 1 mg/kg of patientbody weight or more per day, depending on the factors mentioned above.Preferably the dose ranges from about 10 μg/kg/day to about 100μg/kg/day. The appropriate dosage and treatment regimen can be readilydetermined by the skilled artisan based on a number of factors,including but not limited to the nature of the miR-155 antagonist, theroute of administration, and the subject's state. Efficacy is evaluatedby observing a delay or slowing inflammatory progression.

Example 17—Modulation of miR-155 Target Gene Expression

This example illustrates the modulation of miR-155 target geneexpression in a subject.

A target cell(s) is identified as being in need of modulation of miR-155target gene expression. For example, a target cell(s) is identified byidentifying a subject having, or at risk of developing amyeloproliferative disease such as AML, or as experiencing TLR-mediatedinflammation. Cells such as hematopoietic cells, bone marrow cells,myeloid precursor cells, myeloid cells, macrophages, and the like ofsaid subject are identified as target cells. Target cells are contactedwith an miR-155 antagonist, such as an miR-155 antisense compound. Thesubject is provided daily dose of an miR-155 antagonist, which mightrange from about 0.01 μg/kg to about 1 mg/kg of subject body weight ormore per day, depending on the factors mentioned above. Preferably thedose ranges from about 10 μg/kg/day to about 100 μg/kg/day. Theappropriate dosage and treatment regimen can be readily determined bythe skilled artisan based on a number of factors, including but notlimited to the nature of the miR-155 antagonist, the route ofadministration, and the subject's disease state, or condition. Levels ofmiR-155 target genes such as Cutl1, Arntl, Picalm, Jarid2, PU.1, Csf1r,HIF1α, Sla, Cepbβ, and Bach within the target cell(s) can be determinedbefore and after administration of the miR-155 antagonist.

All patents and publications are herein incorporated by reference intheir entireties to the same extent as if each individual publicationwas specifically and individually indicated to be incorporated byreference.

The invention illustratively described herein suitably can be practicedin the absence of nay element or elements, limitation or limitationsthat is not specifically disclosed herein. The terms and expressionwhich have been employed are used as terms of description and not oflimitation, and there is no intention that the use of such terms andexpressions indicates the exclusion of equivalents of the features shownand described, or portions thereof. It is recognized that variousmodifications are possible within the scope of the invention disclosed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention.

1. (canceled)
 2. A method of modulating the expression of Hif1α gene ina subject: administering to the subject an miR-155 antisense compoundcomprising a modified oligonucleotide consisting of 12 to 30 linkednucleosides in an amount effective to modulate the expression of Hif1αgene, wherein the nucleobase sequence of the modified oligonucleotide iscomplementary to a sequence at least 90% identical to maturemicroRNA-155 (SEQ ID NO: 73), wherein the subject has or is suspected ofsuffering from inflammation or an inflammatory-related condition.
 3. Themethod of claim 2, wherein the nucleobase sequence of the modifiedoligonucleotide has no more than two mismatches to the nucleobasesequence of mature microRNA-155 (SEQ ID NO: 73).
 4. The method of claim2, wherein the modified oligonucleotide comprises a sequence fullycomplementary to the sequence of mature microRNA-155 (SEQ ID NO: 73). 5.The method of claim 2, wherein the modified oligonucleotide comprises atleast 15 contiguous nucleobases of SEQ ID NO:
 74. 6. The method of claim5, wherein the modified oligonucleotide comprises SEQ ID NO:
 74. 7. Themethod of claim 2, wherein the modified oligonucleotide comprises atleast one sugar-modified nucleoside.
 8. The method of claim 7, whereinthe sugar-modified nucleoside comprises a modified sugar selected fromthe group consisting of a bicyclic sugar moiety, a 2′-fluoro sugar, a2′-O-methyl sugar, and a 2′-O-methoxyethyl sugar.
 9. The method of claim2, wherein the inflammation is inflammation arising from amacrophage-induced inflammatory response.
 10. The method of claim 2,wherein the inflammation is mediated by a Toll-like receptor (TLR). 11.The method of claim 10, wherein said TLR is selected from the groupconsisting of TLR2, TLR3, TLR4, and TLR9.
 12. The method of claim 2,wherein the inflammation or inflammatory-related condition is caused bycancer, viral infection or microbial infection.
 13. The method of claim2, wherein the inflammation or inflammatory-related condition isselected from the group consisting of sepsis, septic shock,neurodegeneration, neutrophilic alveolitis, asthma, hepatitis,inflammatory bowel disease, ischemia, glomerulonephritis, rheumatoidarthritis, and Crohn's disease.
 14. The method of claim 2, wherein theadministration comprises parenteral administration.
 15. The method ofclaim 2, comprising administering at least one additional therapy to thesubject in addition to the miR-155 antisense compound.
 16. The method ofclaim 15, wherein the at least one additional therapy comprises animmunosuppressant.
 17. The method of claim 16, wherein theimmunosuppressant is a corticosteroid.
 18. The method of claim 15,wherein the at least one additional therapy comprises a pharmaceuticalagent that enhances the body's immune system.
 19. The method of claim18, wherein the pharmaceutical agent is selected from the groupconsisting of low-dose cyclophosphamide, thymostimulin, vitamins,nutritional supplements, and vaccines.
 20. The method of claim 2,wherein the modified oligonucleotide consists of 15 to 25 linkednucleosides.